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Effect of temperature on biodegradability of surfactants in aquatic microcosm system
~
Pergamon
War. Sci. Tech. Vol. 34, No. 7-8. pp. 61-68,1996. Copyright © 1996 IAWQ. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved. 0273-1223/96 $15'00 + 0'00
PH: S0273-1223(96)00725-1
EFFECT OF TEMPERATURE ON BIODEGRADABILITY OF SURFACTANTS IN AQUATIC MICROCOSM SYSTEM Y. Takamatsu*, o. Nishimura**, Y. Inamori**, R. Sudo*** and M. Matsumura* * Doctoral Program in Agricultural Sciences, University of Tsukuba, J-J-J Tennodai, Tsukuba-shi, lbaraki 305, Japan ** National Institute for Environmental Studies, J6-2 Onogawa. Tsukuba-shi, Ibaraki305, Japan *** Faculty of Engineering, Tohoku University, Aoba-ku, Sendai-shi, Miyagi 980, Japan
ABSTRACT Microcosm systems consisting of producer, decomposer and predator were employed to assess the effect of surfactants (LAS and soap) on an aquatic ecosystem at various temperatures. At all test temperatures (10, 20, 25 and 30°C), stable ecosystems were formed with regard to the biomass and species composition in flasks. In the stationary phase, temperature dependency of ATP was observed and the biodegradation rate of the surfactants in microcosm system at low temperature were slower than that at high temperature. Cyclidium glaucoma, Philodina sp. and Aeolosoma hemprichi as predator were more influenced by surfactants at low temperature. No observed effect concentration (NOEC) of LAS was less than 0.5mg ol- 1 at 10°C. less than 1.5mgol- 1 at 20. 25°C and less than 2.5mgol- 1 at 30°C. NOEC of soap was less than IOmgol-1 at 10°C, less than 30mg ol- 1 at 20, 25°C. It was found that biodegradabilty of surfactants differed with temperature, which changed the effects of surfactants on microorganisms. Copyright © 1996 IAWQ. Published by Elsevier Science Ltd.
KEYWORDS Temperature; surfactant; LAS; soap; biodegradabilty; microcosm; aquatic ecosystem. INTRODUCTION The stability of the water environment in Japan has been improved, but there are many remaining problems to be solved urgently. In particular the direct discharging of gray water is the cause of various water pollution problems. One of the problems is that, great quantities of surfactants in gray water flow directly into the aquatic environments and deteriorate the aquatic ecosystem, even though there is little problem in sewered areas because they are effectively treated by sewage treatment facilities. In the aquatic environment, water temperature changes with the turning of the seasons and the biodegradability and toxicity of surfactants depend on it. To establish an assessment method for surfactants in the environment, it is necessary to resolve the effects of surfactants on the aquatic ecosystem at various temperatures. Environmental assessment of chemical substances has been excuted with EC 50 and LC 50> and an environmental quality standard has been determined by multiplying the value of EC 50 or LC 50 by a safety 61
62
Y. TAKAMATSU
er at.
coeffiCient. However. it is difficult to determine the values of EC so and LC so for the aquatic ecosystem . k'mds 0 f aquatlc '1'Ivmgs, . an d eac h one h as 'Its own value . To ,assess because it IS composed of variOUS environmental effects of surfactant under ecosystem level, it is necessary to analyze the mechamsm of community stability under natural water environment, since natural ecosystems are ~xt~emely co.mplex and are exposed to unpredictable environmental facto~s .. Instead of ~~tural ecosystems It IS convement to use microcosm systems consisting of biotic and non-bIOtic factors ongmated from a natural ecosystem, because they provide biotic simplicity and replication (Bayers 1963~ Cook. 1967; Margalef 19~9; Ka~abata et al., 1978). The microcosm system is comparable with a natural aquatIC ecosystem at baSIC fun~tlOns such as material cycle, energy flow, interaction of microorganisms and so on (Kurihara 198~; Inamon et at., I992!. The microcosm system is considered as an useful environmental assessment met~od 10 thes~ .respects. In this study, the effects of surfactant (LAS and soap) on the aquatic ecosy~tem, and blOdegradab!h~y of surfact~nt in it were examined under various temperature conditions using a mIcrocosm system consIst1Og of bactena, algae and micro animals. EXPERIMENTAL PROCEDURE Microcosm The microcosm system used in this study consisted of four species of bacteria (Bacillus cereus, Pseudomonas putida, Acinetobacter sp. and Coryneform bacteria) as decomposer, one species of ciliate protozoa (Cyclidium glaucoma), two species of rotifers (Philodina sp. and Lepadella sp.) and one species of aquatic oligochaete (Aeolosoma hemprichi) as predator, and a green alga (Chiorella sp.) and a filamentous blue-green alga (Tolypothrix sp.) as producer. This system shows very high reproductiv'ity and reflectivity of natural ecosystem (Shikano et al., 1988; Kurihara 1989). It has been suggested that this small-scale repeatable microcosm can be used as a tool for screening tests on generic ecosystem-level toxicity (Inamori et al., 1992; Sugiura 1992). Surfactants Surfactants tested in this study were LAS (Wako Pure Chemical Industries, Ltd.) and soap (Toho Co.). The sodium Iiner-dodecyl benzene sulfonate standard (C 12), above 99% purity was added as LAS and the sodium fatty acids (C g-C 1g) were added as soap. Initial concentration series of each surfactant were adjusted to 0.5, 1.5,2.5,5.0 and 10.0 mgel- I for LAS and 10,30,50, 100 and 200 mgel- l for soap. LAS was analyzed with HPLC after solid phase extraction by sep-pak C I8 (Waters Co.) and soap analyzed with spectrophotometer after solvent extraction with chloroform. Cultivation The microcosm was cultivated at 10, 20, 25 and 30°C under static conditions with illumination of 2 800 lux (l2UI2D). A 300ml conical flask containing 200 ml of Taub's basal medium (Taub et al., 1964) ~as used and the polypeptone was added to the medium with initial concentration of 50 mgel- I . Surfactants were added to microcosm after 45 days at lOoC, after 16 days at 20, 25 and 30°C from the start of cultivation at which. time all microorganisms in the microcosm system had reached the stationary phase. The numbers' of bactena, protozoa, metazoa. and algae were counted under a microscope. Concentration of LAS and soap were measured at the same tIme. ATP was also measured with a luminometer (Meidensha). RESULTS AND DISCUSSION Growth patterns of microorganisms in the microcosm at various temperatures
~he numbers of each .microorganism under various temperature conditions are shown in Fig. I. At 25°C, the tIme to reach the statIOnary phase was 16 days, and the numbers of microorganisms in the stationary phase 6 were 1.1 x 10 Neml- 1 for bacteria, 6.0 x 10 1 Nem- 1 for C. glaucoma, 1.0 x 10 1 Neml- I for Lepadella sp.,
63
Biodegradability of surfactants in aquatic microcosm system
1.1 X WI N-m1- 1 for Philodina sp., 6 N-ml- I for A. hemprichi, 1.1 x 105 N-ml- I for Chlorella sp., and 8.0 x
102 cm-ml- I for Tolypothrix sp. In the stationary phase the numbers of microorganisms at 10, 20 and 30°C agreed approximately withn those at 25°C except for C. glaucoma which was eliminated from the system after 14 days at 30°C. At 10°C, all microorganisms in the microcosm system grew for 45 days, and then entered to the stationary phase. In other temperature conditions, systems reached the stationary phase after 16 days. Though the time to reach the stationary phase depended on temperature, stable ecosystems consisting of producer, decomposer and predator were formed at all test temperatures, and the difference between these was not recognized with regard to the biomass and species composition.
iz 15
30
45
60
90
75
10
days
10
-.---+-
days
days
20
30
30
20
Philodina sp. Chlorella sp.
Cyclidium glaucoma Aeolosoma hemprichi
Lepadella sp. -0--
Tolypothrix sp.
Bacteria Figure 1. Growth patterns of microorganisms in the microcosm system at various temperatures. 3.00e-7.....--------------
...... ....
2.00e-7
1O'C
20'C
25'C
30'C
temperature Figure 2. Effect of temperature on ATP in the microcosm system at the stationary phase.
Whenever a small quantity of culture at the stationary phase is inoculated to the fresh media, growth curves of each microorganism indicate the same patterns at all test temperatures. Therefore, we concluded that the system was stable, reproducible, and useful for environmental assessment.
Y. TAKAMATSU etal.
es which . 2 ). Th oug h biomass were At each temperature, ATP was compared at the stationary pha.se (FIg. erat~re, . I th same in spite of temperature, ATP decreased WIth the decrease of temp t h' h approximate y e . I all r than that a Ig indicated that the activity of each microorgamsm at ow temperature was sm e temperature. Biodegradation of surfactants in microcosm systems rf b · degraded gradually after addition of surfactants in microcosm systems. With the Su actants were 10 ° 25 0e d 9 days at 300C addition of LAS lO mg-I- I , it took 55 days at woe (Fig.. 3),12 days at 20 e and .. ,an. for biodegradation of LAS. When soap was added to the microcosm, soap scum preCIpItated In the bottom of the flask with reaction between soap and metallic ions. So both supernatant a~d soap ~~u~5wer~ ;~a!~z~d. With the addition of soap 200 mg-l- l , it took 15 days at woe (Fig. 3),. 6 ays at ' . . an or biodegradation of the soap in the supernatant. While soap of the whole mIcroco~m (contaI~Ing soap scum) was biodegraded only about 40% of the additional quantIty at any temperatur~, It was consIdered that soap scum which formed with reaction between soap and metallic io~s was h~dly bIodegraded. It was found that the biodegradability of LAS and soap differed with temperature In the nucrocosm system.
12 ~----------_ _----,
[SOAPl
250 . - - - - - - - - - - - -
-,
2'mA......--.........---..-_----"--...._----..-___
~~/::::I:o=_ -O------o--.Q......;;=-_-..;~I--a 10
-
-0--
----+-
Q5mg'I- 1 5.0mg - H
20
-----
30 days
40
I.5l1l! . I-I
~
50
2.5l1l! . I-I
10.Ong -1- 1
IO.Omg·I-1 (addition to sleri!ized water)
-
60
-0--
---~
50
IQng • j-I --03Ong'I-1 lOOmg • j-I --i!r-200lll! . I-I 200mg'l-l (addition to steri!ized water)
i
60
50mg'I- 1
supernatant
Figure 3. Biodegradation of surfactants in microcosm system at
woe.
Effect of LAS on microorganisms Numbers of each microorganisms after 2 days and 14 days from addition of LAS were compared with the control system. l
C. glaucoma. At LAS of 0.5 mg-l- the number of C. glaucoma was kept at the same number as the control system at all test temperatures. At LAS of 1.5 mgel- l , its number decreased from 2.5 x 102 N-ml-] to 9
I N-ml- for 2 days at lOoe, but the number slowly increased again. At LAS of 2.5 mg-I-I, its number decreasedI for 2 days at 25°e, but the number slowly increased again. With the addition of LAS more than 5.0 mg-I- , C. glaucoma was eliminated from the system at all temperatures (Fig. 4). Philodina sp. With the addition of LAS less than 2.5 mg-I- I, the number of Philodina sp. was kept at the same number as the control at all temperatures. At LAS of 5.0 and 10.0 mg-I- I, the numbers decreased for 2 days after addition at all test temperatures, but slowly increased again until reaching the same number as the control. With the addition of LAS 10.0 mg-l-] at lOoe, the number decreased as the day went on and was not recovered. It was considered that LAS was biodegraded slowly at lOoe, and even after 14 days residual LAS affected Philodina sp. (Fig. 4).
Biodegradability of surfactants in aquatic microcosm system
65
Lepadella sp. With the addition of LAS less than 2.5 mgel- I , the number of Lepadella sp. was kept at the same number as the control at all test temperatures. At LAS of 5.0 mgel- I , its number decreased for 2 days at 10°C, but slowly increased again. At any temperature except 10°C, the numbers were not different from the number of the control. At LAS of 10 mgel- I , its numbers decreased for 2 days but then slowly increased at all test temperatures except 10°C. A. hemprichi. At LAS of 0.5 mgel- I , the numbers of A. hemprichi were kept at the same number as the
control at all test temperatures. At LAS of 1.5 and 2.5 mgel- l , the numbers decreased for 2 days at 10°C, but slowly increased again. At LAS of 5.0 mgel- l , though the numbers decreased for 2 days at all test temperatures, they slowly increased again. At LAS of 10.0 mgel- 1, A. hemprichi was eliminated from the sytem at any temperature except 30°C, the number increased again after decrease until reaching the same number as the control (Fig. 4).
Chiorella sp. The numbers Chiorella sp. were kept at the same number as the control at any temperatures and additional quantities. The effect of LAS on Chiorella sp. was not recognized. Toiypothrix sp. With the addition of less than 2.5 mgel- l of LAS, the effect of LAS on ToLypothrix sp. was not recognized at all test temperatures. Over 2.5 mgel- l of LAS, ToLypothrix sp. was eliminated from the system. ~--------i
2 days after addition 1--------.
I 14 days after addition I
(Philodina sp.)
10 2
'*"' 10 °
S
,,, ,, , ,,, ,, ,,
,,
,, , ,,,
~
(Philodina sp.)
to 2
~---.---~==:;==~----r-------; ,,
Control
e
, : Ll...
'-
10 1
I;I
: Ll....
0.5 1.5 2.5 5.0 Concentration of LAS (mg' I-I)
W
10
'*"'
~
e
(Aeolosoma hemprichi )
10°
rJ>
....S fa er0
L..l... Control
,, ,,, ,,, ,, ,,, , L..l...
.~
Control
1.5 2.5 5.0 0.5 Concentration of LAS (mg' 1-1)
10
10°
~
Control
:l
( Cyclitlium glaucoma) t0 3
0
Z
,-----.------.----.-----,----,----,
L..l..
,, ,,, ,,
L..l...
l......!...
,,, , ,,, ,, , ,,
:
2.5 1.5 5.0 0.5 Concentration of LAS (mg' 1-1)
,, ,,, ,,, ,, ,,, ,
L..l..
, ,,, , ,,, ,, , ,, ,
L..l...
, ,,, ,,, , ,, , ,,, L..l...
.
10
, ,,, , ,,, ,, ,,, ,,
2.5 5.0 0.5 1.5 Concentralion of LAS (mg' I-l)
10
,, ,,
, L....l...
10°
Control
10
,, , ,,
,,, ,, ,, ,,
10 1
0.5 1.5 2.5 5.0 Concentration of LAS (mg . 1-1)
,, ,,, ,,
,,, , ,,, ,,, ,, ,,,
(Cyclidium glaUf:oma ) 10 3 10 2
Control
,
,,, ,,,
( Aeolosoma hemprichi ) 10 1
rJ>
8 .....S
,,, ,,
, ~
,,
,,,
,,,
5.0 2.5 1.5 0.5 Concentration of LAS (mg' 1-1)
,, ,, ,, , ,,, 10
I -------•
lOt
~ 20t
D
25t
D
30t
Figure 4 Comparison of number of microorganisms at 2 days and 14 days after addition of LAS.
Bacteria. With the addition of 2.5 mgel- 1 LAS, the number of bacteria was kept at the same l~vel as that of the control at all test temperatures. At LAS of 5.0 and 10.0 mgel- 1, the number increased rangmg from 10 to
Y. TAKAM ATSU eta!.
102 times higher than that of the control for 2 days after addition. It was consid ered that the increase of bacteria was due to the decrease of predators including C. glaucoma. From the results mentioned above, no observed effect concentration (NOEC ) of LAS ~as less than 0.5 mg-I- I oe I at lo ,ess th an I .5 mg-I,1 at 20 and 25°e and less than 2.5 mg-I- I at 30°C, respectIvely. It was found that . I microorganisms in the system were more influenced by LAS at low temperature than hIgh t~mperatu~e. t was recognized that the biodegradation rate at lower temperature was much slower and mlcroo rgams ms were exposed to LAS for a much longer time under that condition. Effect of soap on microorganisms The number of each microorganism 2 days and 14 days after additio n of soap were compa red with the control system. C. galucoma. With the addition of soap (10 mg-I- I), the number of C. glaucoma agreed with the.nu mber of the control system at all test temperatures. With the addition of soap (more than 30. mg-I- I), Its ~umb~r decreased for 2 days after the addition at all test temperatures, but the numbe r slowly mcrea sed agam untIl reaching the same number as the control. With the addition of soap (200 mg-l- I), at 100e, the number decreased during the experiment.
Philodina sp. and Lepadella sp. With the addition of soap (more than 30 mg-I- I), these numbe rs slowly increased at all temperatures. The reason for the increase in Philodina sp. and Lepad ella sp. was thought to be due to their predation of the increased bacteria which utilize organi c compounds contai ned in soap. A. hemprichi. Witht the additon of soap (less than 100 mg-I- I ), the numbe rs agreed with the numbe rs of the control at 20, 25 and 30°C. Witht the addition of soap (more than 100 mg-I,I) at 10°C, A. hempr ichi was eliminated from the system. Chlorella sp. No effect of soap on Chlorella sp. was observed under any conditions. Tolypothrix sp. With the addition of soap (less than 30 mg-l- I ), the numbers agreed with the numbe r of the control at all test temperatures. With the addition of soap (more than 50 mg-I- I ), the numbe r slowly decreased, and it was estimated that the growth of Tolypothrix sp. covere d by soap scum was inhibited by light limitation. Bacteria. With the addition of soap (100 mg-I- I ), the numbers were kept at the same level as the control at all test temperatures. At soap of 200 mg-l- I , the number increased and became 10 times higher than that of the control after 2 days from addition. It was considered that the increa se of bacter ia was due to both the decrease of predation pressure by C. galucoma and the utilization of organic compo unds contai ned in soap. From the results mentioned above no observed effect concentration (NOEC ) of soap was less than 10 mg-l- I at IDoe and less than 30 mg-l- I at 20°C and 30°C. It was found that microo rganis ms in the system were more influenced by soap at low temperature that at high temperature because the biodeg radatio n rate at lower temperatures was much slower and microorganisms were expose d to soap for much longer time. Time course of ATP in microcosm systems The ATP of the control was constant during the experiment. With the addition of LAS (less than 2.5 mg-I- I), ATP kept at the same level of the control at 25°C (Fig. 5). When LAS was added to be 5.0 and 10.0 mg-I,I, ATP decre.ased, and the damage to microorganisms such as Lepadella sp. and Philodina sp. were observed under a mIcroscope. At 10, 20 and 30oe, time courses of ATP were approximately the same as those of 25°e. It was proved tha~ ATP reflected the condition of microorganis ms in the microc osm system which could not be evaluated WIth the numbers of microorganisms.
Biodegradability of surfactants in aquatic microcosm system
67
With the addition of soap (less than 10 mg-I- I ), ATP was kept at the same level as the control at 25°C (Fig. 5). When soap was added to be ranging from 30 to 200 mg-I- I , ATP increased. It was suggested that increase of ATP was caused by re-growth of Lepadella sp. and Philodina sp. predating the increased bacteria which utilize organic compounds contained in soap. At 10, 20 and 30°C, time courses of ATP were approximately the same as those of 25°C. 5.0e-7
5.0e-7
LAS
4.0e-7 '::'
.....
"0
e
'-"
~
Soap
4.0e-7 -'-
3.0e-7
3.0e-7
"0
2.0e-7
Ei '-" 2.0e-7
1.0e-7
<
O.Oe+O
~
0
5
days
10
15
---.- control
--0-
1.5mg· 1-1
- - - - 2.5mg '1- 1 ............- 1O.Omg . 1-1
--1::.-
5.0mg . I-I
1.0e-7 O.Oe+O
0
5
days
______ control
10
--0-
- - - - 50mg . J-l 200mg'I-1
15 30mg' I-I
---{:,- l00mg . 1-1
~
Figure 5. Time course of ATP in microcosm system at 25°C.
Evaluation of effects of LAS and soap using microcosm test Evaluations of the effect of LAS and soap using the microcosm test were carried out to classify the assessment level into four classes such as assessment A, B, C and D, assessment A: no observed effect concentration; assessment B: concentration which system recovers; assessment C: concentration which one or more species of predators, producers and decomposers can survive; assessment D: concentration at which system breaks down. Assessment ATP was defined as no observed effect concentration with measurement of ATP. Concentrations of assessment A for both LAS and soap increased with increase of temperature (Table I). It was found that the biodegradability of surfactants differed with temperature, which changed the effects of surfactants on microorganisms. This result suggested that temperature should be one of the most important factors for the environmental assessment. It was considered that the safety concentration for the aquatic ecosystem was that of assessment A. Assessment ATP was similar to assessment B, and it was assumed that these concentrations were the limiting values for the aquatic ecosystem to be able to accept surfactant without serious damage, the microcosm test is useful for assessment on the ecosystem level containing material cycle, energy-flow and interaction of microorganisms which could not be obtained with a single-culture experiment. A natural aquatic ecosystem test using natural water, in which natural microbiota exist has low reproductivity, compared with the microcosm test. We conclude that the microcosm test is applicable for the assessment of the effect of chemical substances on aquatic ecosystem. Table 1. Evaluations of effects of surfactants using microcosm test LAS Assessments A B
C D
A1P
SOAP
(mg' )-1)
Culture temperature 25"C 30"C 20"C 10"C 0.5 1.5 10.0
1.5 1.5 10.0
1.5 2.5 10.0
(mg' I-I)
Culture temperature 10"C 20"C 25"C 30"C
2.5
10
30
30
30 30
30
2.5 10.0
30
30
>200 >200 30
>200 >200 30
> 200 >200 30
>200 >200 30
> 10.0 > 10.0 > 10.0 > 10.0 2.5 2.5 2.5 2.5
Y. TAKAMATSU eral.
68
CONCLUSIONS The purpose of this study was to assess the effect of temperature on the biodegradability of surfactants in the environment using an aquatic flask-size microcosm, and the following results were obtained. At all test temperatures (10, 20 and 30°C), stable ecosystems consisting of producer, decomposer and predator were formed with regard to the biomass and species composition in microcosm. i)
ii) In the stationary phase, though biomasses of all test temperatures were approximately the same, the ATP of low temperature was lower than that of high temperature, which indicated that the activities of each microorganism at low temperature were less than those at high temperature. iii) Biodegradation rate of surfactants in microcosm system at low temperature was slower than that at high temperature. Soap scum which was formed by reaction between soap and metallic ions was hardly biodegraded. iv) Predator Cyclidium glaucoma, Philodina sp. and Aeolosoma hemprichi were more influemced by surfactants (especillay lAS) at low temperature. v) No observed effect concentration (NOEC) of LAS was less than 0.5 mg-I- I at 10°C, less than 1.5 mg-I- I at 20. 25°C and less than 2.5 mg-I- I at 30°C. NOEC of soap was less than 10 mg-I- I at 10°C, less than 30 mg-I- 1 at 20, 25°C and 30°C. vi) It was found that the biodegradability of surfactants differed with temperature, which changed the effects of surfactants on microorganisms. vii) It was suggested that temperature should be considered as one of the most important factors for the environmental assessment. REFERENCES Beyers, R. 1. (1963). The metabolism of twelve aquatic laboratory microecosystems. Ecol. Monogr. 33, 281-306. Cooke, G. D. (1967). The pattern of autotrophic succession in laboratory microcosms. Bioscience, 17, 717-722. Margalef, R. (1969). Diversity and stability: A practical proposal and a model of interdependence. Brookhaven Symp. BioI. 22,25• 37. Kawabata, Z. and Kurihara, Y. (1978). Computer simulation study on the nature of the steady state of the aquatic microcosm. Sci. Rep. Tohiku Univ. 37, 205-218. Inamori, Y., Murakami, K., Sudo, R., Kurihara, Y. and Tanaka, N. (1992). Environmental assessment method for field release of . genetically engineered microorganisms using microcosm systems. Wat. Sci. Tech., 26(9-11), 2161-2164. KUrihara, :. (1~89). Interaction and stability of microbial communities in experimental model systems. Recent Advances in Microbial Ecology. 11-20. Shikano, S. and. Kuri.hara, Y. (1988). Aanlysis of factors controlling responses of an aquatic microcosm to organic loading, HydroblOlogla, 251-257. Sugiuara, K. (1992). A multispecies laboratory microcosm for screening ecotoxicological impacts of h . I E ' Chern., 11,1217-1226. c emlca s, nVlron.
7"'
I
IOXICO.
Taub, F. B. and Dollar, A.. M. (1964). A Chlorella-Daphnia food chain study: the design of a compatible chemically defined culture medIUm, Llrnnol. Oceanogr., 9, 61-74.
Pergamon
War. Sci. Tech. Vol. 34, No. 7-8. pp. 61-68,1996. Copyright © 1996 IAWQ. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved. 0273-1223/96 $15'00 + 0'00
PH: S0273-1223(96)00725-1
EFFECT OF TEMPERATURE ON BIODEGRADABILITY OF SURFACTANTS IN AQUATIC MICROCOSM SYSTEM Y. Takamatsu*, o. Nishimura**, Y. Inamori**, R. Sudo*** and M. Matsumura* * Doctoral Program in Agricultural Sciences, University of Tsukuba, J-J-J Tennodai, Tsukuba-shi, lbaraki 305, Japan ** National Institute for Environmental Studies, J6-2 Onogawa. Tsukuba-shi, Ibaraki305, Japan *** Faculty of Engineering, Tohoku University, Aoba-ku, Sendai-shi, Miyagi 980, Japan
ABSTRACT Microcosm systems consisting of producer, decomposer and predator were employed to assess the effect of surfactants (LAS and soap) on an aquatic ecosystem at various temperatures. At all test temperatures (10, 20, 25 and 30°C), stable ecosystems were formed with regard to the biomass and species composition in flasks. In the stationary phase, temperature dependency of ATP was observed and the biodegradation rate of the surfactants in microcosm system at low temperature were slower than that at high temperature. Cyclidium glaucoma, Philodina sp. and Aeolosoma hemprichi as predator were more influenced by surfactants at low temperature. No observed effect concentration (NOEC) of LAS was less than 0.5mg ol- 1 at 10°C. less than 1.5mgol- 1 at 20. 25°C and less than 2.5mgol- 1 at 30°C. NOEC of soap was less than IOmgol-1 at 10°C, less than 30mg ol- 1 at 20, 25°C. It was found that biodegradabilty of surfactants differed with temperature, which changed the effects of surfactants on microorganisms. Copyright © 1996 IAWQ. Published by Elsevier Science Ltd.
KEYWORDS Temperature; surfactant; LAS; soap; biodegradabilty; microcosm; aquatic ecosystem. INTRODUCTION The stability of the water environment in Japan has been improved, but there are many remaining problems to be solved urgently. In particular the direct discharging of gray water is the cause of various water pollution problems. One of the problems is that, great quantities of surfactants in gray water flow directly into the aquatic environments and deteriorate the aquatic ecosystem, even though there is little problem in sewered areas because they are effectively treated by sewage treatment facilities. In the aquatic environment, water temperature changes with the turning of the seasons and the biodegradability and toxicity of surfactants depend on it. To establish an assessment method for surfactants in the environment, it is necessary to resolve the effects of surfactants on the aquatic ecosystem at various temperatures. Environmental assessment of chemical substances has been excuted with EC 50 and LC 50> and an environmental quality standard has been determined by multiplying the value of EC 50 or LC 50 by a safety 61
62
Y. TAKAMATSU
er at.
coeffiCient. However. it is difficult to determine the values of EC so and LC so for the aquatic ecosystem . k'mds 0 f aquatlc '1'Ivmgs, . an d eac h one h as 'Its own value . To ,assess because it IS composed of variOUS environmental effects of surfactant under ecosystem level, it is necessary to analyze the mechamsm of community stability under natural water environment, since natural ecosystems are ~xt~emely co.mplex and are exposed to unpredictable environmental facto~s .. Instead of ~~tural ecosystems It IS convement to use microcosm systems consisting of biotic and non-bIOtic factors ongmated from a natural ecosystem, because they provide biotic simplicity and replication (Bayers 1963~ Cook. 1967; Margalef 19~9; Ka~abata et al., 1978). The microcosm system is comparable with a natural aquatIC ecosystem at baSIC fun~tlOns such as material cycle, energy flow, interaction of microorganisms and so on (Kurihara 198~; Inamon et at., I992!. The microcosm system is considered as an useful environmental assessment met~od 10 thes~ .respects. In this study, the effects of surfactant (LAS and soap) on the aquatic ecosy~tem, and blOdegradab!h~y of surfact~nt in it were examined under various temperature conditions using a mIcrocosm system consIst1Og of bactena, algae and micro animals. EXPERIMENTAL PROCEDURE Microcosm The microcosm system used in this study consisted of four species of bacteria (Bacillus cereus, Pseudomonas putida, Acinetobacter sp. and Coryneform bacteria) as decomposer, one species of ciliate protozoa (Cyclidium glaucoma), two species of rotifers (Philodina sp. and Lepadella sp.) and one species of aquatic oligochaete (Aeolosoma hemprichi) as predator, and a green alga (Chiorella sp.) and a filamentous blue-green alga (Tolypothrix sp.) as producer. This system shows very high reproductiv'ity and reflectivity of natural ecosystem (Shikano et al., 1988; Kurihara 1989). It has been suggested that this small-scale repeatable microcosm can be used as a tool for screening tests on generic ecosystem-level toxicity (Inamori et al., 1992; Sugiura 1992). Surfactants Surfactants tested in this study were LAS (Wako Pure Chemical Industries, Ltd.) and soap (Toho Co.). The sodium Iiner-dodecyl benzene sulfonate standard (C 12), above 99% purity was added as LAS and the sodium fatty acids (C g-C 1g) were added as soap. Initial concentration series of each surfactant were adjusted to 0.5, 1.5,2.5,5.0 and 10.0 mgel- I for LAS and 10,30,50, 100 and 200 mgel- l for soap. LAS was analyzed with HPLC after solid phase extraction by sep-pak C I8 (Waters Co.) and soap analyzed with spectrophotometer after solvent extraction with chloroform. Cultivation The microcosm was cultivated at 10, 20, 25 and 30°C under static conditions with illumination of 2 800 lux (l2UI2D). A 300ml conical flask containing 200 ml of Taub's basal medium (Taub et al., 1964) ~as used and the polypeptone was added to the medium with initial concentration of 50 mgel- I . Surfactants were added to microcosm after 45 days at lOoC, after 16 days at 20, 25 and 30°C from the start of cultivation at which. time all microorganisms in the microcosm system had reached the stationary phase. The numbers' of bactena, protozoa, metazoa. and algae were counted under a microscope. Concentration of LAS and soap were measured at the same tIme. ATP was also measured with a luminometer (Meidensha). RESULTS AND DISCUSSION Growth patterns of microorganisms in the microcosm at various temperatures
~he numbers of each .microorganism under various temperature conditions are shown in Fig. I. At 25°C, the tIme to reach the statIOnary phase was 16 days, and the numbers of microorganisms in the stationary phase 6 were 1.1 x 10 Neml- 1 for bacteria, 6.0 x 10 1 Nem- 1 for C. glaucoma, 1.0 x 10 1 Neml- I for Lepadella sp.,
63
Biodegradability of surfactants in aquatic microcosm system
1.1 X WI N-m1- 1 for Philodina sp., 6 N-ml- I for A. hemprichi, 1.1 x 105 N-ml- I for Chlorella sp., and 8.0 x
102 cm-ml- I for Tolypothrix sp. In the stationary phase the numbers of microorganisms at 10, 20 and 30°C agreed approximately withn those at 25°C except for C. glaucoma which was eliminated from the system after 14 days at 30°C. At 10°C, all microorganisms in the microcosm system grew for 45 days, and then entered to the stationary phase. In other temperature conditions, systems reached the stationary phase after 16 days. Though the time to reach the stationary phase depended on temperature, stable ecosystems consisting of producer, decomposer and predator were formed at all test temperatures, and the difference between these was not recognized with regard to the biomass and species composition.
iz 15
30
45
60
90
75
10
days
10
-.---+-
days
days
20
30
30
20
Philodina sp. Chlorella sp.
Cyclidium glaucoma Aeolosoma hemprichi
Lepadella sp. -0--
Tolypothrix sp.
Bacteria Figure 1. Growth patterns of microorganisms in the microcosm system at various temperatures. 3.00e-7.....--------------
...... ....
2.00e-7
1O'C
20'C
25'C
30'C
temperature Figure 2. Effect of temperature on ATP in the microcosm system at the stationary phase.
Whenever a small quantity of culture at the stationary phase is inoculated to the fresh media, growth curves of each microorganism indicate the same patterns at all test temperatures. Therefore, we concluded that the system was stable, reproducible, and useful for environmental assessment.
Y. TAKAMATSU etal.
es which . 2 ). Th oug h biomass were At each temperature, ATP was compared at the stationary pha.se (FIg. erat~re, . I th same in spite of temperature, ATP decreased WIth the decrease of temp t h' h approximate y e . I all r than that a Ig indicated that the activity of each microorgamsm at ow temperature was sm e temperature. Biodegradation of surfactants in microcosm systems rf b · degraded gradually after addition of surfactants in microcosm systems. With the Su actants were 10 ° 25 0e d 9 days at 300C addition of LAS lO mg-I- I , it took 55 days at woe (Fig.. 3),12 days at 20 e and .. ,an. for biodegradation of LAS. When soap was added to the microcosm, soap scum preCIpItated In the bottom of the flask with reaction between soap and metallic ions. So both supernatant a~d soap ~~u~5wer~ ;~a!~z~d. With the addition of soap 200 mg-l- l , it took 15 days at woe (Fig. 3),. 6 ays at ' . . an or biodegradation of the soap in the supernatant. While soap of the whole mIcroco~m (contaI~Ing soap scum) was biodegraded only about 40% of the additional quantIty at any temperatur~, It was consIdered that soap scum which formed with reaction between soap and metallic io~s was h~dly bIodegraded. It was found that the biodegradability of LAS and soap differed with temperature In the nucrocosm system.
12 ~----------_ _----,
[SOAPl
250 . - - - - - - - - - - - -
-,
2'mA......--.........---..-_----"--...._----..-___
~~/::::I:o=_ -O------o--.Q......;;=-_-..;~I--a 10
-
-0--
----+-
Q5mg'I- 1 5.0mg - H
20
-----
30 days
40
I.5l1l! . I-I
~
50
2.5l1l! . I-I
10.Ong -1- 1
IO.Omg·I-1 (addition to sleri!ized water)
-
60
-0--
---~
50
IQng • j-I --03Ong'I-1 lOOmg • j-I --i!r-200lll! . I-I 200mg'l-l (addition to steri!ized water)
i
60
50mg'I- 1
supernatant
Figure 3. Biodegradation of surfactants in microcosm system at
woe.
Effect of LAS on microorganisms Numbers of each microorganisms after 2 days and 14 days from addition of LAS were compared with the control system. l
C. glaucoma. At LAS of 0.5 mg-l- the number of C. glaucoma was kept at the same number as the control system at all test temperatures. At LAS of 1.5 mgel- l , its number decreased from 2.5 x 102 N-ml-] to 9
I N-ml- for 2 days at lOoe, but the number slowly increased again. At LAS of 2.5 mg-I-I, its number decreasedI for 2 days at 25°e, but the number slowly increased again. With the addition of LAS more than 5.0 mg-I- , C. glaucoma was eliminated from the system at all temperatures (Fig. 4). Philodina sp. With the addition of LAS less than 2.5 mg-I- I, the number of Philodina sp. was kept at the same number as the control at all temperatures. At LAS of 5.0 and 10.0 mg-I- I, the numbers decreased for 2 days after addition at all test temperatures, but slowly increased again until reaching the same number as the control. With the addition of LAS 10.0 mg-l-] at lOoe, the number decreased as the day went on and was not recovered. It was considered that LAS was biodegraded slowly at lOoe, and even after 14 days residual LAS affected Philodina sp. (Fig. 4).
Biodegradability of surfactants in aquatic microcosm system
65
Lepadella sp. With the addition of LAS less than 2.5 mgel- I , the number of Lepadella sp. was kept at the same number as the control at all test temperatures. At LAS of 5.0 mgel- I , its number decreased for 2 days at 10°C, but slowly increased again. At any temperature except 10°C, the numbers were not different from the number of the control. At LAS of 10 mgel- I , its numbers decreased for 2 days but then slowly increased at all test temperatures except 10°C. A. hemprichi. At LAS of 0.5 mgel- I , the numbers of A. hemprichi were kept at the same number as the
control at all test temperatures. At LAS of 1.5 and 2.5 mgel- l , the numbers decreased for 2 days at 10°C, but slowly increased again. At LAS of 5.0 mgel- l , though the numbers decreased for 2 days at all test temperatures, they slowly increased again. At LAS of 10.0 mgel- 1, A. hemprichi was eliminated from the sytem at any temperature except 30°C, the number increased again after decrease until reaching the same number as the control (Fig. 4).
Chiorella sp. The numbers Chiorella sp. were kept at the same number as the control at any temperatures and additional quantities. The effect of LAS on Chiorella sp. was not recognized. Toiypothrix sp. With the addition of less than 2.5 mgel- l of LAS, the effect of LAS on ToLypothrix sp. was not recognized at all test temperatures. Over 2.5 mgel- l of LAS, ToLypothrix sp. was eliminated from the system. ~--------i
2 days after addition 1--------.
I 14 days after addition I
(Philodina sp.)
10 2
'*"' 10 °
S
,,, ,, , ,,, ,, ,,
,,
,, , ,,,
~
(Philodina sp.)
to 2
~---.---~==:;==~----r-------; ,,
Control
e
, : Ll...
'-
10 1
I;I
: Ll....
0.5 1.5 2.5 5.0 Concentration of LAS (mg' I-I)
W
10
'*"'
~
e
(Aeolosoma hemprichi )
10°
rJ>
....S fa er0
L..l... Control
,, ,,, ,,, ,, ,,, , L..l...
.~
Control
1.5 2.5 5.0 0.5 Concentration of LAS (mg' 1-1)
10
10°
~
Control
:l
( Cyclitlium glaucoma) t0 3
0
Z
,-----.------.----.-----,----,----,
L..l..
,, ,,, ,,
L..l...
l......!...
,,, , ,,, ,, , ,,
:
2.5 1.5 5.0 0.5 Concentration of LAS (mg' 1-1)
,, ,,, ,,, ,, ,,, ,
L..l..
, ,,, , ,,, ,, , ,, ,
L..l...
, ,,, ,,, , ,, , ,,, L..l...
.
10
, ,,, , ,,, ,, ,,, ,,
2.5 5.0 0.5 1.5 Concentralion of LAS (mg' I-l)
10
,, ,,
, L....l...
10°
Control
10
,, , ,,
,,, ,, ,, ,,
10 1
0.5 1.5 2.5 5.0 Concentration of LAS (mg . 1-1)
,, ,,, ,,
,,, , ,,, ,,, ,, ,,,
(Cyclidium glaUf:oma ) 10 3 10 2
Control
,
,,, ,,,
( Aeolosoma hemprichi ) 10 1
rJ>
8 .....S
,,, ,,
, ~
,,
,,,
,,,
5.0 2.5 1.5 0.5 Concentration of LAS (mg' 1-1)
,, ,, ,, , ,,, 10
I -------•
lOt
~ 20t
D
25t
D
30t
Figure 4 Comparison of number of microorganisms at 2 days and 14 days after addition of LAS.
Bacteria. With the addition of 2.5 mgel- 1 LAS, the number of bacteria was kept at the same l~vel as that of the control at all test temperatures. At LAS of 5.0 and 10.0 mgel- 1, the number increased rangmg from 10 to
Y. TAKAM ATSU eta!.
102 times higher than that of the control for 2 days after addition. It was consid ered that the increase of bacteria was due to the decrease of predators including C. glaucoma. From the results mentioned above, no observed effect concentration (NOEC ) of LAS ~as less than 0.5 mg-I- I oe I at lo ,ess th an I .5 mg-I,1 at 20 and 25°e and less than 2.5 mg-I- I at 30°C, respectIvely. It was found that . I microorganisms in the system were more influenced by LAS at low temperature than hIgh t~mperatu~e. t was recognized that the biodegradation rate at lower temperature was much slower and mlcroo rgams ms were exposed to LAS for a much longer time under that condition. Effect of soap on microorganisms The number of each microorganism 2 days and 14 days after additio n of soap were compa red with the control system. C. galucoma. With the addition of soap (10 mg-I- I), the number of C. glaucoma agreed with the.nu mber of the control system at all test temperatures. With the addition of soap (more than 30. mg-I- I), Its ~umb~r decreased for 2 days after the addition at all test temperatures, but the numbe r slowly mcrea sed agam untIl reaching the same number as the control. With the addition of soap (200 mg-l- I), at 100e, the number decreased during the experiment.
Philodina sp. and Lepadella sp. With the addition of soap (more than 30 mg-I- I), these numbe rs slowly increased at all temperatures. The reason for the increase in Philodina sp. and Lepad ella sp. was thought to be due to their predation of the increased bacteria which utilize organi c compounds contai ned in soap. A. hemprichi. Witht the additon of soap (less than 100 mg-I- I ), the numbe rs agreed with the numbe rs of the control at 20, 25 and 30°C. Witht the addition of soap (more than 100 mg-I,I) at 10°C, A. hempr ichi was eliminated from the system. Chlorella sp. No effect of soap on Chlorella sp. was observed under any conditions. Tolypothrix sp. With the addition of soap (less than 30 mg-l- I ), the numbers agreed with the numbe r of the control at all test temperatures. With the addition of soap (more than 50 mg-I- I ), the numbe r slowly decreased, and it was estimated that the growth of Tolypothrix sp. covere d by soap scum was inhibited by light limitation. Bacteria. With the addition of soap (100 mg-I- I ), the numbers were kept at the same level as the control at all test temperatures. At soap of 200 mg-l- I , the number increased and became 10 times higher than that of the control after 2 days from addition. It was considered that the increa se of bacter ia was due to both the decrease of predation pressure by C. galucoma and the utilization of organic compo unds contai ned in soap. From the results mentioned above no observed effect concentration (NOEC ) of soap was less than 10 mg-l- I at IDoe and less than 30 mg-l- I at 20°C and 30°C. It was found that microo rganis ms in the system were more influenced by soap at low temperature that at high temperature because the biodeg radatio n rate at lower temperatures was much slower and microorganisms were expose d to soap for much longer time. Time course of ATP in microcosm systems The ATP of the control was constant during the experiment. With the addition of LAS (less than 2.5 mg-I- I), ATP kept at the same level of the control at 25°C (Fig. 5). When LAS was added to be 5.0 and 10.0 mg-I,I, ATP decre.ased, and the damage to microorganisms such as Lepadella sp. and Philodina sp. were observed under a mIcroscope. At 10, 20 and 30oe, time courses of ATP were approximately the same as those of 25°e. It was proved tha~ ATP reflected the condition of microorganis ms in the microc osm system which could not be evaluated WIth the numbers of microorganisms.
Biodegradability of surfactants in aquatic microcosm system
67
With the addition of soap (less than 10 mg-I- I ), ATP was kept at the same level as the control at 25°C (Fig. 5). When soap was added to be ranging from 30 to 200 mg-I- I , ATP increased. It was suggested that increase of ATP was caused by re-growth of Lepadella sp. and Philodina sp. predating the increased bacteria which utilize organic compounds contained in soap. At 10, 20 and 30°C, time courses of ATP were approximately the same as those of 25°C. 5.0e-7
5.0e-7
LAS
4.0e-7 '::'
.....
"0
e
'-"
~
Soap
4.0e-7 -'-
3.0e-7
3.0e-7
"0
2.0e-7
Ei '-" 2.0e-7
1.0e-7
<
O.Oe+O
~
0
5
days
10
15
---.- control
--0-
1.5mg· 1-1
- - - - 2.5mg '1- 1 ............- 1O.Omg . 1-1
--1::.-
5.0mg . I-I
1.0e-7 O.Oe+O
0
5
days
______ control
10
--0-
- - - - 50mg . J-l 200mg'I-1
15 30mg' I-I
---{:,- l00mg . 1-1
~
Figure 5. Time course of ATP in microcosm system at 25°C.
Evaluation of effects of LAS and soap using microcosm test Evaluations of the effect of LAS and soap using the microcosm test were carried out to classify the assessment level into four classes such as assessment A, B, C and D, assessment A: no observed effect concentration; assessment B: concentration which system recovers; assessment C: concentration which one or more species of predators, producers and decomposers can survive; assessment D: concentration at which system breaks down. Assessment ATP was defined as no observed effect concentration with measurement of ATP. Concentrations of assessment A for both LAS and soap increased with increase of temperature (Table I). It was found that the biodegradability of surfactants differed with temperature, which changed the effects of surfactants on microorganisms. This result suggested that temperature should be one of the most important factors for the environmental assessment. It was considered that the safety concentration for the aquatic ecosystem was that of assessment A. Assessment ATP was similar to assessment B, and it was assumed that these concentrations were the limiting values for the aquatic ecosystem to be able to accept surfactant without serious damage, the microcosm test is useful for assessment on the ecosystem level containing material cycle, energy-flow and interaction of microorganisms which could not be obtained with a single-culture experiment. A natural aquatic ecosystem test using natural water, in which natural microbiota exist has low reproductivity, compared with the microcosm test. We conclude that the microcosm test is applicable for the assessment of the effect of chemical substances on aquatic ecosystem. Table 1. Evaluations of effects of surfactants using microcosm test LAS Assessments A B
C D
A1P
SOAP
(mg' )-1)
Culture temperature 25"C 30"C 20"C 10"C 0.5 1.5 10.0
1.5 1.5 10.0
1.5 2.5 10.0
(mg' I-I)
Culture temperature 10"C 20"C 25"C 30"C
2.5
10
30
30
30 30
30
2.5 10.0
30
30
>200 >200 30
>200 >200 30
> 200 >200 30
>200 >200 30
> 10.0 > 10.0 > 10.0 > 10.0 2.5 2.5 2.5 2.5
Y. TAKAMATSU eral.
68
CONCLUSIONS The purpose of this study was to assess the effect of temperature on the biodegradability of surfactants in the environment using an aquatic flask-size microcosm, and the following results were obtained. At all test temperatures (10, 20 and 30°C), stable ecosystems consisting of producer, decomposer and predator were formed with regard to the biomass and species composition in microcosm. i)
ii) In the stationary phase, though biomasses of all test temperatures were approximately the same, the ATP of low temperature was lower than that of high temperature, which indicated that the activities of each microorganism at low temperature were less than those at high temperature. iii) Biodegradation rate of surfactants in microcosm system at low temperature was slower than that at high temperature. Soap scum which was formed by reaction between soap and metallic ions was hardly biodegraded. iv) Predator Cyclidium glaucoma, Philodina sp. and Aeolosoma hemprichi were more influemced by surfactants (especillay lAS) at low temperature. v) No observed effect concentration (NOEC) of LAS was less than 0.5 mg-I- I at 10°C, less than 1.5 mg-I- I at 20. 25°C and less than 2.5 mg-I- I at 30°C. NOEC of soap was less than 10 mg-I- I at 10°C, less than 30 mg-I- 1 at 20, 25°C and 30°C. vi) It was found that the biodegradability of surfactants differed with temperature, which changed the effects of surfactants on microorganisms. vii) It was suggested that temperature should be considered as one of the most important factors for the environmental assessment. REFERENCES Beyers, R. 1. (1963). The metabolism of twelve aquatic laboratory microecosystems. Ecol. Monogr. 33, 281-306. Cooke, G. D. (1967). The pattern of autotrophic succession in laboratory microcosms. Bioscience, 17, 717-722. Margalef, R. (1969). Diversity and stability: A practical proposal and a model of interdependence. Brookhaven Symp. BioI. 22,25• 37. Kawabata, Z. and Kurihara, Y. (1978). Computer simulation study on the nature of the steady state of the aquatic microcosm. Sci. Rep. Tohiku Univ. 37, 205-218. Inamori, Y., Murakami, K., Sudo, R., Kurihara, Y. and Tanaka, N. (1992). Environmental assessment method for field release of . genetically engineered microorganisms using microcosm systems. Wat. Sci. Tech., 26(9-11), 2161-2164. KUrihara, :. (1~89). Interaction and stability of microbial communities in experimental model systems. Recent Advances in Microbial Ecology. 11-20. Shikano, S. and. Kuri.hara, Y. (1988). Aanlysis of factors controlling responses of an aquatic microcosm to organic loading, HydroblOlogla, 251-257. Sugiuara, K. (1992). A multispecies laboratory microcosm for screening ecotoxicological impacts of h . I E ' Chern., 11,1217-1226. c emlca s, nVlron.
7"'
I
IOXICO.
Taub, F. B. and Dollar, A.. M. (1964). A Chlorella-Daphnia food chain study: the design of a compatible chemically defined culture medIUm, Llrnnol. Oceanogr., 9, 61-74.