L-Glutamic acid, N-coco acyl derivs., monosodium salts

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Experimental test result performed according to the OECD test guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 7.5, 15, 30, 60 and 120 mg/l
- Sampling method: Samples for analytical determination was collected from four corners and center of the aquarium for each test concentrations send for chemical analysis
- Sample storage conditions before analysis: Samples were immediately analysed after sampling

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test chemical was prepared by dissolving 1250 mg of test chemical in 5000 ml of RO water. After stirring the stock solution was filtered and analytically detected. The final solubility value obtained after analytical detection was 226.81 mg/l. The remaining test solutions were prepared by dilution from the above stock solution. Test chemical concentrations used for the study were 0, 7.5, 15, 30, 60 and 120 mg/l, respectively.
- Controls: RO water as control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra Fish
- Length at study initiation (length definition, mean, range and SD): 1.5 cm
- Housing:The fishes were kept in a static tank in tap water passed through reverse osmosis system, under natural conditions along with proper feed and aeration.
- Duration of housing: 7 days
- Photoperiod: 16 hour light and 8 hour darkness

ACCLIMATION
- Acclimation period: Fishes, to be used in this study, are kept in the test water for seven days immediately before testing
- Acclimation conditions (same as test or not): same test conditions
Photoperiod: 16 hour light and 8 hours dark
Temperature range 22.6°C
pH range 7.9
DO range 8.1 mg/L
Feeding Daily once but not given atleast 48 hours before study
Aeration Continuous
Mortality (%): 0%
- Diet : Standard brand feed, once daily

FEEDING DURING TEST
- Food type: Feeding was not provided during test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

22.6°C

pH:

7.9

Dissolved oxygen:

8.1 mg/l

Nominal and measured concentrations:

Test chemical concentrations used for the study were 0, 7.5, 15, 30, 60 and 120 mg/l, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquarium
- Material, size, headspace, fill volume: 7 liters of test vessel filled with 4 liter of water
- Aeration: Aeration in test vessels was provided 1 day before the start of experiment
- No. of organisms per vessel: 7 test organisms per vessel
- No. of vessels per concentration (replicates): one vessel per concentrations
- No. of vessels per control (replicates): 1 vessel per control
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: 7 fishes /4 L

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8-hour darkness

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: RO water obatined from the RO water filter systems

OTHER TEST CONDITIONS
- Adjustment of pH: Not adjusted
- Photoperiod: 16 hours light and 8 hours dark

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: Not applicable

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

No toxic effect was observed against fish at the limit test mg/L of water solubility under exposure conditions.
After the 96 hours of exposure, there was no mortality in the control group, and the dissolved oxygen concentration was 87.14% of the air saturation value.

Results with reference substance (positive control):

Not applicable

Sublethal observations / clinical signs:

Measured Concentration-At 0and96thhour

 

S.No	Nominal Conc.	0Hr		96Hr
		Absorbance (mean)	Measuredconc.	Absorbance (mean)	Measuredconc.
1	blank	0.00	0.00	0.00	0.00
2	7.5	0.07	17.47	-	-
3	15.00	0.08	19.66	-	-
4	30.00	0.15	36.03	0.14	32.44
5	60.00	0.28	66.47	0.13	31.76
6	120.00	0.55	129.02	0.41	96.90

 

Measured ConcentrationbeforeRenewalandAfterRenewal-24Hr

 

S.No.	Nominal Conc.	Absorbance (mean)	Measuredconc.	Absorbance (mean)	Measuredconc.
	blank	24Hr(BeforeRenewal )		24Hr(AfterRenewal)
1	7.5	0.13	30.09	0.08	19.77
2	15.00	0.14	32.71	0.13	30.31
3	30.00	0.19	45.97	0.21	50.58
4	60.00	0.28	67.07	0.41	96.80
5	120.00	0.45	106.92	0.71	167.76

 

 

 

Measured ConcentrationbeforeRenewalandAfterRenewal-48Hr

 

S.No.	Nominal Conc.	Absorbance (mean)	Measuredconc.	Absorbance (mean)	Measuredconc.
	blank	48Hr(BeforeRenewal )		48Hr(AfterRenewal)
1	7.5	0.00	-1.01	0.02	5.81
2	15.00	0.00	-1.36	0.06	14.02
3	30.00	0.03	6.82	0.13	31.50
4	60.00	0.18	42.76	0.25	59.91
5	120.00	0.24	56.18	0.51	120.83

 

Measured ConcentrationbeforeRenewalandAfterRenewal-72Hr

 

S.No.	Nominal Conc.	Absorbance (mean)	Measuredconc.	Absorbance (mean)	Measuredconc.
	blank	72Hr(BeforeRenewal )		72Hr(AfterRenewal)
1	7.5	0.01	3.65	0.06	13.23
2	15.00	0.03	7.09	0.09	20.23
3	30.00	0.07	17.79	0.15	35.30
4	60.00	0.13	31.90	0.26	60.25
5	120.00	0.24	56.65	0.41	98.32

MORTALITYANDVISIBLESYMPTOMS

 

Sr.No	Test concentrations(mg/L)	2.5 Hr	5.5H r	24 Hr	48 Hr	72 Hr	96 Hr	Cumulative Mortality	PercentInhibitio n
1	Control	-	-	-	-	-	-	-	-
2	7.5	-	-	-	-	-	-	-	-
3	15.00	-	-	-	-	-	-	-	-
4	30.00	-	-	-	-	-	-	-	-
5	60.00	-	-	-	-	-	-	-	-
6	120.00	-	-	-	-	-	-	-	-

 

 

 

 

Sr.No	Test concentrations(mg/L )	2.5Hr	5.5Hr	24Hr	48Hr	72Hr	96Hr
1	Control	NS7	NS7	NS7	NS7SC7	NS7	NS7
2	7.5	NS7	NS7	NS7	NS7SC7	NS7	NS7
3	15.00	NS7	NS7	NS7	NS7SC7	NS7	NS7
4	30.00	NS7	NS7	NS7	NS7SC7	NS7	NS7
5	60.00	NS7	NS7	NS7	NS7SC7	M7	M7
6	120.00	NS7	NS7	NS7	NS7SC7	M7	M7
	Anyother symptoms

 

EXPLANATIONOFSYMPTOMS:

 Numberbehindsymbolfor symptom =numberofaffectedfish

 A:apathy,NR:Narcotic-likestate,C:Swimmingnearwatersurface,M:Slowmovementscomparedtocontrol, NS: No abnormal symptoms,SC: Schooling effect, L: Loss of equilibrium, SM: Shimmingmovement,H1:Hyperactivity,H2:HypoactivityB:Bendingbehavior,F:Finrot,IS:Invertswimming,SB:Swimming     behavior,     RF:     Respiratory     Function,     P:     Pigmentation,     E:Exophthalmos

 ANNEX2:pH,TEMPERATURE, ANDDISSOLVEOXYGEN(Before Renewal)

 

TestConcentration(mg/L)	pH
	24Hours	48Hours	72Hours	96Hours
Control	7.7                7	.7	7.7	7.8
7.5	7.8	7.8	7.7	7.8
15.00	7.8	7.8	7.8	7.9
30.00	7.8	7.8	7.7	7.8
60.00	7.7	7.7	7.6	7.7
120.00	7.7	7.6	7.6	7.7

 

 

TestConcentration(mg/L)	Temperature °C
	24Hours	48Hours	72Hours	96Hours
Control	22	21.9	23	22
7.5	22	21.9	22	22
15.00	22	21.9	22	22
30.00	21	21.9	22	22
60.00	21	21.9	22	22
120.00	22	21.9	22	22

 

 

	TestConcentration(mg/L)	DissolvedOxygen
		24Hours	48Hours	72Hours	96Hours
	Control	7.1	7.5	7.4	8.0
	7.5	6.5	7.1	6.5	6.9
	15.00	6.7	6.6	6.3	6.7
	30.00	6.1	6.5	5.3	6.1
	60.00	5.5	6.2	5.0	4.1
	120.00	5.1	6.0	3.5	3.8

ANNEX3:pH,TEMPERATURE, ANDDISSOLVEOXYGEN(After renewal)

Test Concentration(mg/L) pH   24Hours   48Hours   72Hours   96Hours   Control 7.9 7.8 7.9 -   7.5 7.9 7.9 8.0 -   15.00 7.9 7.9 8.0 -   30.00 7.9 7.9 7.9 -   60.00 7.9 7.9 8.0 -   120.00 7.9 7.9 7.9 -

 

f TestConcentration(mg/L)	Temperature °C
	24Hours	48Hours	72Hours	96Hours
Control	21	22.3	22.4	-
7.5	21	22.3	22.4	-
15.00	21	22.3	22.4	-
30.00	22	22.3	22.4	-
60.00	22	22.3	22.4	-
120.00	22	22.3	22.4	-

 

 

TestConcentration(mg/L)	DissolvedOxygen
	24Hours	48Hours	72Hours	96Hours
Control	7.1	8.5	8.8	-
7.5	7.7	8.9	8.4	-
15.00	7.7	8.7	8.2	-
30.00	7.5	8.9	7.7	-
60.00	7.5	8.7	7.8	-
120.00	7.0	8.6	7.2	-

Validity criteria fulfilled:

yes

Conclusions:

Based on measured concentrations, the median lethal concentration [LC50 (96 h)] for test chemical on Danio rerio (Zebra Fish) was determined to be >120mg/L (nominal conc.).

Executive summary:

An acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical on Zebra fish (Danio rerio). The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average length of 1.5 cm was used as a test organism for the study. Test fishes were kept in a static tank in tap water passed through reverse osmosis system, under natural conditions along with proper feed and aeration. During the housing period, test fishes were fed once daily with standard brand fed. Aeration in test vessels was provided till 1 day before the start of the experiment. The test chemical was prepared by dissolving 1250 mg of test chemical in 5000 ml of RO water. After stirring the stock solution was filtered and analytically detected. The final solubility value obtained after analytical detection was 226.81 mg/l. The remaining test solutions were prepared by dilution from the above stock solution. Test chemical concentrations were analytically determined by UV-VIS spectrophotometer. Test chemical conc. used for the study were 0, 7.5, 15, 30, 60 and 120 mg/l, respectively. Total 7 fishes were exposed to test chemical in a 7 lit of glass aquarium containing 4000 ml of potable water. The test vessels were placed in a room at a temperature of 22.6°C, pH of 7.9 and DO of 78.1 mg/l and under a photoperiod of 16:8 hr light: dark conditions, respectively. At regular intervals of 24 hours vessels were observed for any behavioural changes and mortality along with that the DO, pH and temperature was measured. Test chemical was sampled from test vessel for analytical determinations at 0 hour and 96 hours. All the test concentrations were analytical determined and at 96 hours of the exposure durations which were maintained in the range of 80 -120%. Therefore, the analysis of the results was based on nominal concentration. After the 96 hours of exposure, there was no mortality in the control group, and the dissolved oxygen concentration was 87.14% (i.e. above 60%) of the air saturation value throughout the exposure period. Thus, the study fulfilling the validity criterion as mentioned in the OECD TG 203. On the basis of effect of test chemical on mortality of the test organism, the 96 hr median lethal concentration [LC50 (96 h)] for test chemical on Danio rerio (Zebra Fish) was determined to be >120 mg/L (nomnal conc.). Thus, based on LC50 value, test chemical was considered as non-toxic to aquatic fishes and hence, considered to be 'not classified' category as per the CLP classification criteria.

Description of key information

An acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical on Zebra fish (Danio rerio). The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average length of 1.5 cm was used as a test organism for the study. Test fishes were kept in a static tank in tap water passed through reverse osmosis system, under natural conditions along with proper feed and aeration. During the housing period, test fishes were fed once daily with standard brand fed. Aeration in test vessels was provided till 1 day before the start of the experiment. The test chemical was prepared by dissolving 1250 mg of test chemical in 5000 ml of RO water. After stirring the stock solution was filtered and analytically detected. The final solubility value obtained after analytical detection was 226.81 mg/l. The remaining test solutions were prepared by dilution from the above stock solution. Test chemical concentrations were analytically determined by UV-VIS spectrophotometer. Test chemical conc. used for the study were 0, 7.5, 15, 30, 60 and 120 mg/l, respectively. Total 7 fishes were exposed to test chemical in a 7 lit of glass aquarium containing 4000 ml of potable water. The test vessels were placed in a room at a temperature of 22.6°C, pH of 7.9 and DO of 78.1 mg/l and under a photoperiod of 16:8 hr light: dark conditions, respectively. At regular intervals of 24 hours vessels were observed for any behavioural changes and mortality along with that the DO, pH and temperature was measured. Test chemical was sampled from test vessel for analytical determinations at 0 hour and 96 hours. All the test concentrations were analytical determined and at 96 hours of the exposure durations which were maintained in the range of 80 -120%. Therefore, the analysis of the results was based on nominal concentration. After the 96 hours of exposure, there was no mortality in the control group, and the dissolved oxygen concentration was 87.14% (i.e. above 60%) of the air saturation value throughout the exposure period. Thus, the study fulfilling the validity criterion as mentioned in the OECD TG 203. On the basis of effect of test chemical on mortality of the test organism, the 96 hr median lethal concentration [LC50 (96 h)] for test chemical on Danio rerio (Zebra Fish) was determined to be >120 mg/L (nomnal conc.). Thus, based on LC50 value, test chemical was considered as non-toxic to aquatic fishes and hence, considered to be 'not classified' category as per the CLP classification criteria.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

120 mg/L

Additional information

Experimental study of the test chemical and supporting weight of evidence studies for its read across substance were reviewed for the short term toxicity to fish endpoint which are as summarized below:

In an experimental study report, an acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical on Zebra fish (Danio rerio). The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average length of 1.5 cm was used as a test organism for the study. Test fishes were kept in a static tank in tap water passed through reverse osmosis system, under natural conditions along with proper feed and aeration. During the housing period, test fishes were fed once daily with standard brand fed. Aeration in test vessels was provided till 1 day before the start of the experiment. The test chemical was prepared by dissolving 1250 mg of test chemical in 5000 ml of RO water. After stirring the stock solution was filtered and analytically detected. The final solubility value obtained after analytical detection was 226.81 mg/l. The remaining test solutions were prepared by dilution from the above stock solution. Test chemical concentrations were analytically determined by UV-VIS spectrophotometer. Test chemical conc. used for the study were 0, 7.5, 15, 30, 60 and 120 mg/l, respectively. Total 7 fishes were exposed to test chemical in a 7 lit of glass aquarium containing 4000 ml of potable water. The test vessels were placed in a room at a temperature of 22.6°C, pH of 7.9 and DO of 78.1 mg/l and under a photoperiod of 16:8 hr light: dark conditions, respectively. At regular intervals of 24 hours vessels were observed for any behavioural changes and mortality along with that the DO, pH and temperature was measured. Test chemical was sampled from test vessel for analytical determinations at 0 hour and 96 hours. All the test concentrations were analytical determined and at 96 hours of the exposure durations which were maintained in the range of 80 -120%. Therefore, the analysis of the results was based on nominal concentration. After the 96 hours of exposure, there was no mortality in the control group, and the dissolved oxygen concentration was 87.14% (i.e. above 60%) of the air saturation value throughout the exposure period. Thus, the study fulfilling the validity criterion as mentioned in the OECD TG 203. On the basis of effect of test chemical on mortality of the test organism, the 96 hr median lethal concentration [LC50 (96 h)] for test chemical on Danio rerio (Zebra Fish) was determined to be >120 mg/L (nomnal conc.). Thus, based on LC50 value, test chemical was considered as non-toxic to aquatic fishes and hence, considered to be 'not classified' category as per the CLP classification criteria.

In a supporting weight of evidence study from secondary source, short term fish toxicity was conducted for 96 hrs for assessing the effect of test chemical. The test was performed following the principles of the OECD Guideline 203 (Fish, Acute Toxicity Test).Oryzias latipes (Japanese rice fish) of length 2.38 cm (S.D. = 0.11 cm) and 0.12 g (S.D. = 0.02g) weight obtained from National Institute of Environmental Research (Environmental Research Complex, Kyungseo-dong, Seo-gu, Incheon, Korea) was used as a test organism. Upon receipt, suitable male and female fish, at a ratio of 3 to 2 respectively, without any visible abnormalities and of similar length were selected and placed in a 50 L glass breeding chamber containing maintenance water. Approximately 50 fish (±10%) were bred. Eggs were harvested from the breeding chambers and placed in hatching chambers. After hatching, the fry were placed in a breeding chamber at 23±2°C and bred. Approximately 30% of the holding water is exchanged once a week. The holding room was artificially illuminated; 16 hours light, 8hours dark. Fish were fed Brine Shrimp (Ocean Star International, Inc., U.S.A.) in the morning and Top Meal (Jaeilfeed Co., Korea) in the afternoon, approximately 3% of their body weight daily, except on Sunday when they were fasted. Prior to initiation, fish without any visible abnormalities and of similar length were selected and acclimated in maintenance water for 10 days. During the acclimation period, the water temperature and dissolved oxygen concentration in maintenance water were maintained at 22.1–23.8°C and 86.4–96.1%, respectively. The room was artificially illuminated with 16 hours of light and 8 hours of dark. The fish were fed Brine Shrimp eggs (Ocean Star International, Inc., U.S.A.) in the morning and Top meal in the afternoon, at an amount of approximately 3% of their body weight, once daily except on Sunday when they were fasted. Mortalities were observed from 48 hours after initiation of acclimation. During the seven days prior to exposure, mortalities of a batch of fish were less than 5% of populations, so a batch of fish was used. No food during the 24 hours period immediately prior to exposure. Public tap water was filtered and irradiated by ultraviolet light and used as maintenance and dilution water. The request amounts (active ingredient: 0.998 g) of the test substance were measured using an electronic balance. For preparation of stock and test solutions, the required amounts of the test chemical were added to test chamber filled with dilution water. For the range finding study, a stock solution of 100 mg/L of the test substance was prepared using dilution water. This stock solution was diluted to concentrations of 10 and 1 mg/L of the test substance with dilution water. In definitive study, 100 mg/L test solution of the test substance was prepared. Each of 3 L test solution per test chamber was prepared, and the dilution water was used for the control group. Thus, limit test was performed using 100 mg/l of test chemical concentration. Test chemical concentrations were determined analytically. The concentrations of test chemical were analyzed using HPLC in all test solutions at the beginning (0 hour) and at the end (96 hours) of the study. The samples were taken from mid-layer of 100 mg/L test solutions and centrifuged at 3000 rpm for 5 minutes. Duplicate samples from the top layer of each batch of test solutions diluted within the concentration range of calibration samples and analyzed. Also, duplicate samples from mid-layer of control group were centrifuged at 3000 rpm for 5 minutes and analyzed. Total 10 fishes/conc. were exposed to the test chemical in a test chamber for 96 hrs. Test conditions during the study include hardness of 61 mg/l as CaCO3, temperature of 23.1 to 23.8°C, pH 7.33 to 7.53 and dissolved oxygen of 7.09 to 7.64 mg/l under a photoperiod of 16 hour light, 8 hour dark cycle in a fluorescent lighting. Temperature and oxygen levels were maintained. All experiments including the control were performed in replicate. Mortality of the test organisms were noted. Copper (II) Sulfate was used as a positive control. The 96 hr LC50 value of the reference substance Copper (II) Sulfate was determined to be 0.31 mg/l and it was within the historical control range (mean±2 SD: 0.24–0.48 mg/L). At the end of the test, total length and weight of the control fish were 2.38±0.11 cm and 0.12±0.02 g (mean ± SD), respectively. During the exposure period, the water temperature, dissolved oxygen concentration, and pH of test solution were 23.1–23.8°C, 7.09–7.64 mg/L (converted at air saturation value: 84.5–91.1%) and 7.33–7.53, respectively. These were within the range permitted for the study. The test solutions in the control group were observed for transparency prior to exposure initiation and during the exposure period. The test solutions in the 100 mg/L dosed group were observed for transparency prior to exposure initiation and 24, 48 hours after exposure, while it was shown turbidity at 72 and 96 hours after exposure. All the fish in the control group and the dosed group at 100 mg/L nominal concentration were normal without deaths or abnormal signs. Thus, after an exposure period of 96 hrs, the mortality of the test organism was less than 50% (actual measurement: 0%) in the highest concentration dosed group, so the LC50 at 24, 48, 72, and 96 hours was determined as >100 mg/L, respectively. The concentration of the test substance during the exposure period was within (+/-) 20% of the nominal concentrations. Therefore, all test results were determined as the nominal concentrations. In the control vessel, the mortality of the test organism was not exceed 10% at the end of the exposure, dissolved oxygen concentration was ≥60% of the air saturation value in all test vessels throughout the exposure and analytical measurement of test concentrations were carried out. Thus, validity criteria of the study was fulfilled. On the basis of effect on mortality of the test organism, the 96 hr LC50 value was determined to be > 100 mg/l (nominal conc.) and > 99.47 mg/l (measured conc.), respectively. Thus, based on this value, test chemical was considered as non-toxic to fish and hence, considered to be ‘not classified’ as per the CLP classification criteria.  

 

For the test chemical, another short term fish toxicity was conducted for 96 hrs for assessing the effect of test chemical. Study was performed in accordance with the OECD Guideline 203 (Fish, Acute Toxicity Test) under static conditions. On the basis of the effect of test chemical on mortality of the test fishes, the 96 hrs median lethal concentration (LC50) value was determined to be >21 mg/l. Thus, based on the LC50 value, test chemical was considered as non-toxic to aquatic fishes at environmental relevant concentrations.

On the basis of the above result, test chemical was considered as non-toxic to aquatic fishes and hence, considered to be non toxic as per the CLP classification criteria.