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Toxicity of walnut husk washing waters
War. Res. Vol,26, No. 11, pp. 1503-1506,1992 Printed in Great Britain.All rights reservvd
0043-1354/92$5.00+0.00 Copyright© 1992PergamonPressLtd
TOXICITY OF WALNUT HUSK WASHING WATERS PASCALRADIX, F~NGOXSESEIGLE-MU~NDff, JEAs-Lotns BL~OIT-GUYOD and SERGEKRIVOBOK GEDEXE
(Groupe pour rEtude du Devenir des X6nobiotiques dans rEnvironn©ment), Universit~ J. Fourier (Grenoble I), BP 138, 38243 Meylan, France
(First received February 1991; accepted in revisedform April 1992) AlUxa~--Foflowing a dispute between walnut producers and an anglers association, the toxicity of walnut washings has been determined and followed in a sewer and in a stream in a walnut production area near Grenoble (France) and compared with juglone toxicity. Toxicity of the washings has also been determined after storage at two different temperatures, with and without fight. Juglone was found only to be responsible for a small part of this toxicity.
Key words--Juglans regia L., husk washings, juglone toxicity
INTRODUCTION
MATERIALSAND METHODS
In France and in Europe, the region of Grenoble is a major area for growing the Persian walnut (Juglans regia L.). Before sale of the crop, the husk has to be removed from the nuts. Husking is often accompanied with washing and the washings are, most of the time, thrown directly into the environment. Walnut has been reported to be toxic to a wide variety of organisms including herbaceous and woody plants (Brooks, 1951; Rietveld, 1979). The principal chemical responsible for walnut toxicity is juglone (5-hydroxy-l,4-naphthoquinone) (Davis, 1928). Jugione has been isolated from many plants in the walnut family (Thomson, 1971; Graves et al., 1979; Hedin et al., 1980). It was shown that jugione restricted respiration in plants (Perry, 1967), inhibited mycelial growth (Hedin et al., 1980), had antimicrobial activity (Didry et al., 1 9 8 6 ) a n d affected germination and growth of plants (Rietveld, 1983) and seaweeds (Kessler, 1989). Walnut husks have
Sampling Sampleswere collected at 7 different sites (Fig. 1) while huskingwas going on and stored in polyethylenebottles, at +4°C, in the dark according to Rodier (1984).
pH pH was measured with a Radiometer PHM 82 (Copenhagen).
COD COD was measured according to the French norm NFT 90-101(1988) equivalent to ISO 6060 (1986). The samples t
_~ North
$1
_
ahighcontentofjugloneOVestfalletal.,1961)
~lkm $5------~|T¢ $3__~ It st sower ~ ] ..,
n~ ~' e/t/m l
~noS6~[i
and it was shown that the highest concentration of juglone was obtained during summer (Borazjani, 1982; Borazjani et al., 1985; Tekinkas et al., 1988). A clash occurred recently in Polienas near Grenoble, in the south-eastern part of France, where walnut is a main crop, between an anglers association and walnut producers concerning husk washings and their disposal. The nuts are collected from midSeptember to mid-October. During that period, about 2000 1. of washings per ton of fresh walnuts are thrown into the environment. The purpose of this study was to find out if these washings are toxic and could be dangerous to streams in that area. *Author to whom all correspondence should be addressed, wR26/tl--G
S4
$2 Hus facilities
/ ]| f ~
ditch
1 t ~ , ~
$7
Towards Is~re river Fig. 1. Location of the sampling sites (from S1-$7). Distances: S1--$3= 200 m; $2-S3 = 70 m; $4-$5 = 1500m; $5--$3 ffi 50 m; $3-$6 = 50 m and $6-$7 - 1500m. Terebet stream width: 1-2 m; main ditch width: 3-4 m.
1503
PASCALRADIX et al.
1504
COD pH (mg/l) SI 6.8 605 $2 4.7 5980 $3 5.3 1190 $4 7.2 117 $5 6.8 206 $6 6.6 485 $7 6.8 282 *Details given in Table 3.
Table 1. Analysis of water samples IC~e as Toxicity A r t e m i a Juglone as juglone (I/dilution) (mg/l) (mgfl) -0 0 721 16 810 1.5
-----
Toxicity increase Toxicity*to factor micromycetes -+ 51 + + +
0.6
1.7
2.8
0 0 0 0
0 0 0 0
-----
were acidified, added to a known amount of potassium dichromate, with silver sulfate as oxidation catalyst, with mercury (II) sulfate, to complex chloride ions, and refluxed. Excess dichromate was titrated with a solution of ferrous ammonium (II) sulfate. COD was calculated from the amount of reduced dichromate,
+
+ ++ ++ +
638). The fungitoxicity was tested in Petri dishes (90 mm dia) containing 25 ml of solid malt extract medium (1.5%). Agar-well diffusion assay was carried out by pouring calibrated suspensions of the test strains (10eceils/ml) into the wells. After incubation at 24°C for 48 h, the diameters of the inhibition (in ram) were measured. Each test was carried out in triplicate.
Extraction of juglone flora the samples Extraction was done according to Westfall et ai. (1961): 50rul samples were brought to a pH lower than 5.7 by N/10 H2SO4 and extracted three times with 30 ml of petroleum ether. The extracts were pooled, dried on anhydrous sodium sulfate and evaporated to dryness at 42°C under vacuum. The residues were dissolved in MeOH (10 ml). Yield: 92%. Analysis: juglone was analyzed by HPLC. HPLC was performed on a LC6A Shimadzu equipped with a SP6A detector, a SIL 9A automatic injector and a CR3A integrator. The conditions were as follows: 250 x 5 mm Waters Cm/~bondapak column, I ml min -l, MeOH : H20 75 : 25. Detection 420 nm. Retention time was 5.6 rain and reference was commervial jugione, 90% pure (Sigma Chemical Co., St Louis, Mo., U.S.A.).
Table 1. Toxicity to Artemia (Table 1): for SI a n d $4-$7, the ICs0s could n o t be calculated, even with undiluted washings, as there was less t h a n a 50% death rate.
Toxicity on Artemia salina
Table 3.
Toxicity was evaluated as IC50 on Artemia salina according to Persoone et al. (1980). Obtainment of the nauplii. The cysts came from Creasel Ltd, Deinze, Belgium. Reconstituted sea water was a 35 g/l Instant Ocean solution (Aquarium systems, Sarrebourg, France). pH was adjusted to 8 + 0.5. The solution was filtered on Millipore filters (0.65/~m) before use. Cysts (200mg) were kept at 25°C+ 1 for 24h, under artificial daylight in a flask with 200 ml of sea water and air allowed to bubble gently. After 24h, the hatched nauplli were separated from the remaining cysts and transferred into a second flask, under similar conditions. The nauplii were ready for use after another 24 h. Determination of the lCyo. Preliminary assays and blanks were performed. The samples were diluted and the salt level adjusted to allow the establishment of the ICs0. After a 24 h incubation in the dark, animals that did not move for 10 s were considered as immobilized. The IC50 were also determined after storage of the washings at 20 and 50°C, with and without light (4800 lux/m2). They were calculated using a computer program,
Toxicity ofjuglone on micromycetea Evaluation of antifungal activity was performed according to the agar-well diffusion method (Hufford et al., 1975; Devillers et al., 1989). Test strains came from the University of Grenoble (CMPG: Collection Mycology Pharmacy Grenoble). They included Saccharomyces cerevisiae Hansen (CMPG 2), Aspergillusflavus Link:Fr. (CMPG 24), Botrytis cinerea Pers.:Fr. (CMPG 117), Mucor racemosus Fres. (CMPG 143), Chaetomiam globosum Kunze:Fr. (CMPG 219), Penicilliam aurantiogriseam Dierckx (CMPG 247), Fusarh~m moniliforme Sheldon (CMPG 253), Absidia spinoza Lendner (CMPG 428), Geotrichum candidum Link (CMPG 596) and Phanerochaete chrysosporium Burdsall (CMPG
RESULTS Chemical analysis: C O D , p H a n d juglone concentrations (mg/l) are given for each sample in
The ICs0 o f j u g l o n e was 1.12 rag/1. The influences of light a n d temperature o n the toxicity o f husk washings are given in Table 2. Results of the toxicity to micromycetes are given in
DISCUSSION
pH The samples were acidic (pH 4.5 for $2) b u t due to low ionic strength, the p H returned rapidly to the former values of 5.3 f o r S 3 a n d 6 . 6 forS6. Thus, there should n o t be any detrimental effect o n fauna or flora.
COD The C O D o f the washings reached (5980 mg/l) the high level o f paper industry effluents. F o r a water treatment plant, this level should not exceed 90 mg/l (French norms). It should be noted that the stream already had a high C O D of 117 mg/1 for $4, upstream from Poli6nas village. Thus, it has to be considered that although husking increased the organic load of water, other sources remained u n k n o w n in a rural area without any sewage treatment plant. Table 2. Toxicity to Artemia of sample 2 (husk washings) at two temperatures, with and without light, after storage (IC~ as I/dilution) Days--, 0 I 7 14 30 50
Conditions Dark, 20°C Dark, 50°C Light, 20°C Light, 50°C
810 810 810 810
10 . . . . . . . 174 30 10 . . . .
. . -.
--
Toxicity of walnut husk washing waters
1505
Table 3. Toxicityto l0 strains of micromyccta using the agar-welldiffu~on method CMPG reference Name of strains S! $2 $3 $4 S5 $6 2 Saccharomyces cerevisiae 0 1.8 0 0 0 0 24 Aspergillusflavus ! .0 3.6 1.0 1.0 1.0 1.0 I !7 Botrytis cinerea 0 2.0 1.0 0 0 0 143 Mucor racemosus 0 0 0 0 0 0 219 Chaetomnon giobosum 0 1.7 0 0 0 0 247 Peniciilium aurantiogriseum 0 2.5 0 0 0 0 253 Fusarium moniliforme 2.0 2.5 0 1.8 2.0 2.5 428 Absidia spinosa ! .4 4.0 0 0 1.4 0 596 Geotrichum candidum 0 0 0 0 0 2.5 638 Phanerochaete chrysosporhon 0 3.0 0 0 2.5 2.0
S7 0 1.0 0 0 0 0 2.0 0 0 0
Toxicity of the samples In $2, the toxicity was more than 50-fold what should have been given by the juglone content alone, Thus, other highly toxic compounds come from the washings. Toxicity diminishes rapidly in the sewer and is less than 3-fold the related juglone toxicity in $3, 35 m downstream, although the dilution in the sewer was only about 1/3. The loss of toxicity might be due to pH variations, but more likely to adsorption on suspended matter. N o toxicity was found at any other site downstream. However, it should be pointed out that a higher load of washings might harm fauna and flora.
toxicity or in large amounts, were present but not identified. Pesticide residues should be looked for, as well as copper salts, as large amounts are used in walnut bacteriosis treatment. Moreover, it would be interesting to study the evolution of this toxicity in connection with time, fight and temperature in order to give an adequate solution to walnut producers' improvements of the process. As walnut harvests only last for 1 month and husking is only for a few hours a day, it might be easy, to begin with, to prevent any high toxicity peak by good management of the hours of washing.
Toxicity after storage
Acknowledgement--We thank B. Okeke for valuable contribution to the improvement of this manuscript.
At 50°C, the toxicity of husk washings disappeared in the dark as well as in the daylight in less than 1 day. At 20°C, in the dark, toxicity after 1 day was already very low and disappeared totally on day 7. However, toxicity remained much longer at 20°C in the presence of light. Fast formation of a photoproduct of lower toxicity but of higher stability than the parent products can be hypothesized, Toxicity on micromycetes Sample 2 (husk washings) is clearly the most toxic sample to the 10 micromycete tested strains. Only M. racemosus and G. candidum were unaffected by its toxicity. However, while $3 (end of the sewer) was the second most toxic sample to Artemia, it was not significantly more toxic to micromycetes than any of the other samples, When only strains are considered, C M P G 253 (F. moniliforme) and 24 (A. flavus) are good indicaters of the kind of toxicity. At a lesser level, C M P G 638 (P. chrysosporium) and 428 (A. be considered. Five strains, C M P G 117 (B. cinerea), 219 (C. globosum), griseum) and 596 (G. candidum)are
spinosa) can also 2 (S. cerevisiae), 247 (P. aurantionot very sensitive
to the toxicity of the washings while C M P G 143 (M. racemosus) is completely unaffected. CONCLUSION This study has revealed that walnut washings had a high load of organic matter and a high level of toxicity. Juglone is only responsible for a small part of this toxicity. Other compounds, either of high
Rr~I~nENCF~S Borazjani A. (1982) Occurrence ofjuglone among walnuts and hickories: seasonal variations presence among tissues, translocation and the influence of growth regulations on concentration. Diss. Abstr. Int. 42, 4258. Borazjani A., Graves C. H. and Hedin P. A. (1985) Occurrence ofjuglone in various tissues of pecan. Phytopathoiogy 75, 1419-1421. Brooks M. G. (1951) Effects of black walnut trees and their products on other vegetation. West Va Univ. Agric. Exp. Stn Bull. 347. Collective. Norme FrancoLise (1988) Determination de la demande chimique en oxyg~ne (DCO). NFT 90-101. Davis E. F. (1928) The toxic principle of Juglans nigra as identified with synthetic juglone and its toxic effects on tomato and alfalfa plants. Am. J. Bet. 15, 620. Devillers J., Steiman R. and Seigie-Murandi F. (1989) The usefulness of the agar-weil diffusion method for assessing chemical toxicity to bacteria and fungi. Chemosphere 19, 1693--1700. Didry N., Pinkas M. and Dubreuil L. (1986) Antimicrobial activity of some naphthoquinones found in plant. Ann. Pharm. Ft. 44, 73-78. Graves C. H., Hedin P. A. and Langhans V. E. (1979) A survey of juglone levels among pecan, hickory, and walnut. In Prec. 72ndAnn. Cony. Southeast Growers Assn, Hilton Head, S.C., pp. 113-121. Hedin P. A., CoUum D. H., Langhans V. E. and Graves C . H . (1980) Distribution of juglone and related compounds in pecan and their effect on Fusicladium effusum. J. Agric. Fd Chem. 28, 340-342. Hufford C. D., Funderburk M. J., Morgan J. M. and Robertson L. W. (1975) Two antimicrobial alkaloids from heartwood of Liriodendron tulipifera L. J. pharm. $ci. 64, 789-792.
1506
PASCAL RADIX et al.
Kessler C. T. (1989) Effect of juslone on freshwater algal Rietvcld W. J. (1983) AUelopathic effects of jugione on growth. J. chem. Ecol. 15, 2127-2134. germination and growth of several herbaceous and woody Perry S. F. (1967) Inhibition of respiration by juglone in spe~es. J. chem. Ecol. 9, 295-308. Phaseolus and Lycopersicon. Bull. Torrey Bot. Club 94, Rodier J. (1984) L'analyse de rean. Dunod technique, Paris. 26-30. Tekink~Lq E., Tanrisever A. and Mendilcioglu K. (1988) Persoone G., Vankaecke P., Sorgeloos P. and Claus C. Isolation and annual variation of juglone in walnuts (1980) Intercalibration exercise on a short-term standard (Juglans regia L.). Ege Univ. Ziraat Fak. Derg. 25, toxicity test with Artemia nauplii. Final report ENV. 3 9 6 . 215-225. B(N'). Thomson R. H. (1971) Naturally Occurring Quinones, Rietveld W. J. (1979) Ecological implications of allelopathy 2nd edition, pp. 271-272. Academic Press, New York. in forestry. In Proc. John S. Wright Forestry Conf. (Ecfited Westfall B. A., Russel R. L. and Auyong T. K. (1961) by Holt H. A. and Fischer B. C.), pp. 90-112. Purdue Depressant agent from walnut hulls. Science 134, University, West Lafayette, Ind. 1617.
0043-1354/92$5.00+0.00 Copyright© 1992PergamonPressLtd
TOXICITY OF WALNUT HUSK WASHING WATERS PASCALRADIX, F~NGOXSESEIGLE-MU~NDff, JEAs-Lotns BL~OIT-GUYOD and SERGEKRIVOBOK GEDEXE
(Groupe pour rEtude du Devenir des X6nobiotiques dans rEnvironn©ment), Universit~ J. Fourier (Grenoble I), BP 138, 38243 Meylan, France
(First received February 1991; accepted in revisedform April 1992) AlUxa~--Foflowing a dispute between walnut producers and an anglers association, the toxicity of walnut washings has been determined and followed in a sewer and in a stream in a walnut production area near Grenoble (France) and compared with juglone toxicity. Toxicity of the washings has also been determined after storage at two different temperatures, with and without fight. Juglone was found only to be responsible for a small part of this toxicity.
Key words--Juglans regia L., husk washings, juglone toxicity
INTRODUCTION
MATERIALSAND METHODS
In France and in Europe, the region of Grenoble is a major area for growing the Persian walnut (Juglans regia L.). Before sale of the crop, the husk has to be removed from the nuts. Husking is often accompanied with washing and the washings are, most of the time, thrown directly into the environment. Walnut has been reported to be toxic to a wide variety of organisms including herbaceous and woody plants (Brooks, 1951; Rietveld, 1979). The principal chemical responsible for walnut toxicity is juglone (5-hydroxy-l,4-naphthoquinone) (Davis, 1928). Jugione has been isolated from many plants in the walnut family (Thomson, 1971; Graves et al., 1979; Hedin et al., 1980). It was shown that jugione restricted respiration in plants (Perry, 1967), inhibited mycelial growth (Hedin et al., 1980), had antimicrobial activity (Didry et al., 1 9 8 6 ) a n d affected germination and growth of plants (Rietveld, 1983) and seaweeds (Kessler, 1989). Walnut husks have
Sampling Sampleswere collected at 7 different sites (Fig. 1) while huskingwas going on and stored in polyethylenebottles, at +4°C, in the dark according to Rodier (1984).
pH pH was measured with a Radiometer PHM 82 (Copenhagen).
COD COD was measured according to the French norm NFT 90-101(1988) equivalent to ISO 6060 (1986). The samples t
_~ North
$1
_
ahighcontentofjugloneOVestfalletal.,1961)
~lkm $5------~|T¢ $3__~ It st sower ~ ] ..,
n~ ~' e/t/m l
~noS6~[i
and it was shown that the highest concentration of juglone was obtained during summer (Borazjani, 1982; Borazjani et al., 1985; Tekinkas et al., 1988). A clash occurred recently in Polienas near Grenoble, in the south-eastern part of France, where walnut is a main crop, between an anglers association and walnut producers concerning husk washings and their disposal. The nuts are collected from midSeptember to mid-October. During that period, about 2000 1. of washings per ton of fresh walnuts are thrown into the environment. The purpose of this study was to find out if these washings are toxic and could be dangerous to streams in that area. *Author to whom all correspondence should be addressed, wR26/tl--G
S4
$2 Hus facilities
/ ]| f ~
ditch
1 t ~ , ~
$7
Towards Is~re river Fig. 1. Location of the sampling sites (from S1-$7). Distances: S1--$3= 200 m; $2-S3 = 70 m; $4-$5 = 1500m; $5--$3 ffi 50 m; $3-$6 = 50 m and $6-$7 - 1500m. Terebet stream width: 1-2 m; main ditch width: 3-4 m.
1503
PASCALRADIX et al.
1504
COD pH (mg/l) SI 6.8 605 $2 4.7 5980 $3 5.3 1190 $4 7.2 117 $5 6.8 206 $6 6.6 485 $7 6.8 282 *Details given in Table 3.
Table 1. Analysis of water samples IC~e as Toxicity A r t e m i a Juglone as juglone (I/dilution) (mg/l) (mgfl) -0 0 721 16 810 1.5
-----
Toxicity increase Toxicity*to factor micromycetes -+ 51 + + +
0.6
1.7
2.8
0 0 0 0
0 0 0 0
-----
were acidified, added to a known amount of potassium dichromate, with silver sulfate as oxidation catalyst, with mercury (II) sulfate, to complex chloride ions, and refluxed. Excess dichromate was titrated with a solution of ferrous ammonium (II) sulfate. COD was calculated from the amount of reduced dichromate,
+
+ ++ ++ +
638). The fungitoxicity was tested in Petri dishes (90 mm dia) containing 25 ml of solid malt extract medium (1.5%). Agar-well diffusion assay was carried out by pouring calibrated suspensions of the test strains (10eceils/ml) into the wells. After incubation at 24°C for 48 h, the diameters of the inhibition (in ram) were measured. Each test was carried out in triplicate.
Extraction of juglone flora the samples Extraction was done according to Westfall et ai. (1961): 50rul samples were brought to a pH lower than 5.7 by N/10 H2SO4 and extracted three times with 30 ml of petroleum ether. The extracts were pooled, dried on anhydrous sodium sulfate and evaporated to dryness at 42°C under vacuum. The residues were dissolved in MeOH (10 ml). Yield: 92%. Analysis: juglone was analyzed by HPLC. HPLC was performed on a LC6A Shimadzu equipped with a SP6A detector, a SIL 9A automatic injector and a CR3A integrator. The conditions were as follows: 250 x 5 mm Waters Cm/~bondapak column, I ml min -l, MeOH : H20 75 : 25. Detection 420 nm. Retention time was 5.6 rain and reference was commervial jugione, 90% pure (Sigma Chemical Co., St Louis, Mo., U.S.A.).
Table 1. Toxicity to Artemia (Table 1): for SI a n d $4-$7, the ICs0s could n o t be calculated, even with undiluted washings, as there was less t h a n a 50% death rate.
Toxicity on Artemia salina
Table 3.
Toxicity was evaluated as IC50 on Artemia salina according to Persoone et al. (1980). Obtainment of the nauplii. The cysts came from Creasel Ltd, Deinze, Belgium. Reconstituted sea water was a 35 g/l Instant Ocean solution (Aquarium systems, Sarrebourg, France). pH was adjusted to 8 + 0.5. The solution was filtered on Millipore filters (0.65/~m) before use. Cysts (200mg) were kept at 25°C+ 1 for 24h, under artificial daylight in a flask with 200 ml of sea water and air allowed to bubble gently. After 24h, the hatched nauplli were separated from the remaining cysts and transferred into a second flask, under similar conditions. The nauplii were ready for use after another 24 h. Determination of the lCyo. Preliminary assays and blanks were performed. The samples were diluted and the salt level adjusted to allow the establishment of the ICs0. After a 24 h incubation in the dark, animals that did not move for 10 s were considered as immobilized. The IC50 were also determined after storage of the washings at 20 and 50°C, with and without light (4800 lux/m2). They were calculated using a computer program,
Toxicity ofjuglone on micromycetea Evaluation of antifungal activity was performed according to the agar-well diffusion method (Hufford et al., 1975; Devillers et al., 1989). Test strains came from the University of Grenoble (CMPG: Collection Mycology Pharmacy Grenoble). They included Saccharomyces cerevisiae Hansen (CMPG 2), Aspergillusflavus Link:Fr. (CMPG 24), Botrytis cinerea Pers.:Fr. (CMPG 117), Mucor racemosus Fres. (CMPG 143), Chaetomiam globosum Kunze:Fr. (CMPG 219), Penicilliam aurantiogriseam Dierckx (CMPG 247), Fusarh~m moniliforme Sheldon (CMPG 253), Absidia spinoza Lendner (CMPG 428), Geotrichum candidum Link (CMPG 596) and Phanerochaete chrysosporium Burdsall (CMPG
RESULTS Chemical analysis: C O D , p H a n d juglone concentrations (mg/l) are given for each sample in
The ICs0 o f j u g l o n e was 1.12 rag/1. The influences of light a n d temperature o n the toxicity o f husk washings are given in Table 2. Results of the toxicity to micromycetes are given in
DISCUSSION
pH The samples were acidic (pH 4.5 for $2) b u t due to low ionic strength, the p H returned rapidly to the former values of 5.3 f o r S 3 a n d 6 . 6 forS6. Thus, there should n o t be any detrimental effect o n fauna or flora.
COD The C O D o f the washings reached (5980 mg/l) the high level o f paper industry effluents. F o r a water treatment plant, this level should not exceed 90 mg/l (French norms). It should be noted that the stream already had a high C O D of 117 mg/1 for $4, upstream from Poli6nas village. Thus, it has to be considered that although husking increased the organic load of water, other sources remained u n k n o w n in a rural area without any sewage treatment plant. Table 2. Toxicity to Artemia of sample 2 (husk washings) at two temperatures, with and without light, after storage (IC~ as I/dilution) Days--, 0 I 7 14 30 50
Conditions Dark, 20°C Dark, 50°C Light, 20°C Light, 50°C
810 810 810 810
10 . . . . . . . 174 30 10 . . . .
. . -.
--
Toxicity of walnut husk washing waters
1505
Table 3. Toxicityto l0 strains of micromyccta using the agar-welldiffu~on method CMPG reference Name of strains S! $2 $3 $4 S5 $6 2 Saccharomyces cerevisiae 0 1.8 0 0 0 0 24 Aspergillusflavus ! .0 3.6 1.0 1.0 1.0 1.0 I !7 Botrytis cinerea 0 2.0 1.0 0 0 0 143 Mucor racemosus 0 0 0 0 0 0 219 Chaetomnon giobosum 0 1.7 0 0 0 0 247 Peniciilium aurantiogriseum 0 2.5 0 0 0 0 253 Fusarium moniliforme 2.0 2.5 0 1.8 2.0 2.5 428 Absidia spinosa ! .4 4.0 0 0 1.4 0 596 Geotrichum candidum 0 0 0 0 0 2.5 638 Phanerochaete chrysosporhon 0 3.0 0 0 2.5 2.0
S7 0 1.0 0 0 0 0 2.0 0 0 0
Toxicity of the samples In $2, the toxicity was more than 50-fold what should have been given by the juglone content alone, Thus, other highly toxic compounds come from the washings. Toxicity diminishes rapidly in the sewer and is less than 3-fold the related juglone toxicity in $3, 35 m downstream, although the dilution in the sewer was only about 1/3. The loss of toxicity might be due to pH variations, but more likely to adsorption on suspended matter. N o toxicity was found at any other site downstream. However, it should be pointed out that a higher load of washings might harm fauna and flora.
toxicity or in large amounts, were present but not identified. Pesticide residues should be looked for, as well as copper salts, as large amounts are used in walnut bacteriosis treatment. Moreover, it would be interesting to study the evolution of this toxicity in connection with time, fight and temperature in order to give an adequate solution to walnut producers' improvements of the process. As walnut harvests only last for 1 month and husking is only for a few hours a day, it might be easy, to begin with, to prevent any high toxicity peak by good management of the hours of washing.
Toxicity after storage
Acknowledgement--We thank B. Okeke for valuable contribution to the improvement of this manuscript.
At 50°C, the toxicity of husk washings disappeared in the dark as well as in the daylight in less than 1 day. At 20°C, in the dark, toxicity after 1 day was already very low and disappeared totally on day 7. However, toxicity remained much longer at 20°C in the presence of light. Fast formation of a photoproduct of lower toxicity but of higher stability than the parent products can be hypothesized, Toxicity on micromycetes Sample 2 (husk washings) is clearly the most toxic sample to the 10 micromycete tested strains. Only M. racemosus and G. candidum were unaffected by its toxicity. However, while $3 (end of the sewer) was the second most toxic sample to Artemia, it was not significantly more toxic to micromycetes than any of the other samples, When only strains are considered, C M P G 253 (F. moniliforme) and 24 (A. flavus) are good indicaters of the kind of toxicity. At a lesser level, C M P G 638 (P. chrysosporium) and 428 (A. be considered. Five strains, C M P G 117 (B. cinerea), 219 (C. globosum), griseum) and 596 (G. candidum)are
spinosa) can also 2 (S. cerevisiae), 247 (P. aurantionot very sensitive
to the toxicity of the washings while C M P G 143 (M. racemosus) is completely unaffected. CONCLUSION This study has revealed that walnut washings had a high load of organic matter and a high level of toxicity. Juglone is only responsible for a small part of this toxicity. Other compounds, either of high
Rr~I~nENCF~S Borazjani A. (1982) Occurrence ofjuglone among walnuts and hickories: seasonal variations presence among tissues, translocation and the influence of growth regulations on concentration. Diss. Abstr. Int. 42, 4258. Borazjani A., Graves C. H. and Hedin P. A. (1985) Occurrence ofjuglone in various tissues of pecan. Phytopathoiogy 75, 1419-1421. Brooks M. G. (1951) Effects of black walnut trees and their products on other vegetation. West Va Univ. Agric. Exp. Stn Bull. 347. Collective. Norme FrancoLise (1988) Determination de la demande chimique en oxyg~ne (DCO). NFT 90-101. Davis E. F. (1928) The toxic principle of Juglans nigra as identified with synthetic juglone and its toxic effects on tomato and alfalfa plants. Am. J. Bet. 15, 620. Devillers J., Steiman R. and Seigie-Murandi F. (1989) The usefulness of the agar-weil diffusion method for assessing chemical toxicity to bacteria and fungi. Chemosphere 19, 1693--1700. Didry N., Pinkas M. and Dubreuil L. (1986) Antimicrobial activity of some naphthoquinones found in plant. Ann. Pharm. Ft. 44, 73-78. Graves C. H., Hedin P. A. and Langhans V. E. (1979) A survey of juglone levels among pecan, hickory, and walnut. In Prec. 72ndAnn. Cony. Southeast Growers Assn, Hilton Head, S.C., pp. 113-121. Hedin P. A., CoUum D. H., Langhans V. E. and Graves C . H . (1980) Distribution of juglone and related compounds in pecan and their effect on Fusicladium effusum. J. Agric. Fd Chem. 28, 340-342. Hufford C. D., Funderburk M. J., Morgan J. M. and Robertson L. W. (1975) Two antimicrobial alkaloids from heartwood of Liriodendron tulipifera L. J. pharm. $ci. 64, 789-792.
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Kessler C. T. (1989) Effect of juslone on freshwater algal Rietvcld W. J. (1983) AUelopathic effects of jugione on growth. J. chem. Ecol. 15, 2127-2134. germination and growth of several herbaceous and woody Perry S. F. (1967) Inhibition of respiration by juglone in spe~es. J. chem. Ecol. 9, 295-308. Phaseolus and Lycopersicon. Bull. Torrey Bot. Club 94, Rodier J. (1984) L'analyse de rean. Dunod technique, Paris. 26-30. Tekink~Lq E., Tanrisever A. and Mendilcioglu K. (1988) Persoone G., Vankaecke P., Sorgeloos P. and Claus C. Isolation and annual variation of juglone in walnuts (1980) Intercalibration exercise on a short-term standard (Juglans regia L.). Ege Univ. Ziraat Fak. Derg. 25, toxicity test with Artemia nauplii. Final report ENV. 3 9 6 . 215-225. B(N'). Thomson R. H. (1971) Naturally Occurring Quinones, Rietveld W. J. (1979) Ecological implications of allelopathy 2nd edition, pp. 271-272. Academic Press, New York. in forestry. In Proc. John S. Wright Forestry Conf. (Ecfited Westfall B. A., Russel R. L. and Auyong T. K. (1961) by Holt H. A. and Fischer B. C.), pp. 90-112. Purdue Depressant agent from walnut hulls. Science 134, University, West Lafayette, Ind. 1617.