Reaction mass of sodium hydrogen N-(1-oxooctadecyl)-L-glutamate and sodium hydrogen N-(1-oxohexadecyl)-L-glutamate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 July 2017 to 28 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law)

Version / remarks:

Guidelines for studies on the new chemical substance as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law) 1973, amended 2009 under the reference of YAKUSHOKHATSU No. 1121002, SEIKYOKU No.2 and KANPOKIHATSU No. 021121002 and partially amended 2006 as the joint
ordinance of The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 2-7-1-1

Version / remarks:

The guidelines related to the study reports for the registration application of pesticide (Ref. No. 12-Nousan-8147 on 24 November 2000) & Ref. No.13-Seisan-3986 on 10 October 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Duplicate samples were taken (2 mL) in plastic tubes at the beginning and at the end of the first and last renewal periods from the control and at the applied test concentration levels.
- After sampling, samples were frozen and kept approximately at -20 °C at the Test Facility. One set of the samples was sent to the Test Site for analysis and one set was retained as a back-up at the Test Facility, if required for any confirmatory analyses (discarded after satisfactory results were obtained on the first set of).
- The study was performed as a static renewal test. During this 96-hour study the test substance was renewed four times. Analytical determinations were carried out at the start and the end of the first and of the last renewal cycle. A more frequent analysis was not considered necessary as measured values were close to nominal values.

Vehicle:

no

Remarks:

(dilution water used as test medium)

Details on test solutions:

- Although the test material is well soluble in water, it precipitates in aquarium water. Precipitation was likely to be caused by the "encounter" of test material and dissolved calcium and magnesium content of the aquarium water (i.e. the hardness of the water). In case of filtration used, test material is not present in the "solution" practically. During the analytical method development micelle formation was observed at high concentration stock solution, so the concentration was above the critical micelle concentration (CMC) due to its chemical structure (anionic surfactant). Gel formation is predicted at high concentrations.
- Due to its chemical structure the above mentioned salt precipitation reaction occurs when the test material is placed into the aquarium water. For reasons detailed above the experiment was carried out in a different media, in which the test material could be kept in solution.
- Due to problems with preparation of test material stock solution (opalescent test solution and precipitation when trying to dissolve it in aquarium water) deionised water was used as a test medium instead of aquarium water.
- Prior to treatment, a stock solution with a concentration of 100 mg/L (nominal) was prepared with direct addition of the test material, mixed into the test medium using ultrasonic bath approximately for 5 minutes. The test solutions were prepared by appropriate diluting of this stock solution just before introduction of the fish.
- Because the test material is not stable over the 96h exposure period (based on the analytical method validation) the test was performed under semi-static conditions. The frequency of the water renewal periods was 24 hours. Prior to treatment of each renewal period, test material solutions were prepared by the method described above.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Length at study initiation: 2.6 – 2.9 cm
- Weight at study initiation: 0.18-0.22 g

ACCLIMATION
- Acclimation period: 12 days
- Health during acclimation: Fish were bred in a well-known fish farm, under diseaseand parasite-controlled conditions. Fish were observed during the acclimatisation and test periods, the fish were healthy. There was no mortality of the population for seven days before the test.

FEEDING DURING TEST
- The fish were not fed during the test

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

12 mg/L (as CaCO3)

Test temperature:

20.5 - 22.3 °C

pH:

6.30 – 6.97

Dissolved oxygen:

- The dissolved oxygen concentration was in the range of 80 – 99 % of the air saturation value at the temperature used. Test solutions were aerated during the second renewal period to avoid decreasing of dissolved oxygen concentration below the accepted level.

Nominal and measured concentrations:

Nominal: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L
Measured: 5.9, 12.3, 25.9, 51.9 and 104.7 mg/L
As the measured concentrations deviated not more than 20 per cent from the nominal in all cases, the biological results are based on the nominal test material concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: An aquarium
- Material, size, headspace, fill volume: 5 L test solution
- Renewal rate of test solution: every 24 hours
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates):1
- Biomass loading rate: The loading of the test aquaria was less than 1.0 g fish/L test solution at the start of the experiment.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water was used as a test medium instead of aquarium water due to precipitation.
- Intervals of water quality measurement: The water temperature, oxygen concentration and pH were measured at the start and at the end of renewal periods in each test aquarium (where the fish were alive).

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours darkness

EFFECT PARAMETERS MEASURED: The observations of fish were carried out in the following intervals: at 3, 6, 24, 48, 72 and 96 hours. Mortality and any sub-lethal effect were determined in each observation period.

TEST CONCENTRATIONS
Range finding study:
- Test concentrations: 0.1, 1, 10 and 100 mg/L (nominal)
Four fish were exposed to each concentration of the test item plus a control, for 96 hours under semi-static conditions. The frequency of the water renewal periods was 24 hours.
- Results used to determine the conditions for the definitive study: yes the choice of the test concentrations was made on the basis of the results of the preliminary range-finding test.

Definitive test:
- Because toxic response was observed at the highest examined concentration level during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.0) and one control group were tested in the definitive study in a semi-static system. The frequency of the water renewal periods was 24 hours.
- The nominal concentrations of test item used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

LC50

Remarks:

and 24 h

Effect conc.:

17.85 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: (95 % conf. limits: 12.01 – 24.75 mg/L)

Key result

Duration:

96 h

Dose descriptor:

LC50

Remarks:

and 72 h

Effect conc.:

14.21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: (95 % conf. limits: 10.25 – 30.45 mg/L)

Key result

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

VALIDITY
The achievable validity criteria were within acceptable limits and therefore the study can be considered as valid:
- Constant conditions should be maintained in this procedure: Please see tables below.
- The mortality in the controls should not exceed 10 % at the end of the test (or one fish if less than ten are used). In case of limit test, no mortality should occur: No mortality occurred in the control group during the test.
- The dissolved oxygen concentration (throughout the test) has to be at least 60 % of air saturation value: The dissolved oxygen concentrations were between 80 and 99 % in all test groups.
- The concentration of the test item should be at least 80 % of the nominal concentration throughout the test. If the deviation from the nominal concentration is greater than 20%, results will be based on the measured concentration: The percentage of nominal concentration measured in the analytical test samples was 91.4 % to 104.8 %.

MORTALITY DATA
- Cumulative mortality data from the exposure of zebrafish to the test material during the definitive test are given in the table below.

OBSERVATIONS
- Behaviour of fish was found as normal in the control group and at the lowest tested concentration level of 6.25 mg/L (nominal).
- In the test material group of 12.5 mg/L (nominal) localization close to the surface of the water was observed 72 hours after the start of the experiment, while decreased activity, localisation at the bottom of the aquaria or close to the surface of the water, fast motility of operculum and immobility was noted at the test group of 25.0 mg/L (nominal) during the first 6 hours of the experiment and all fish died 24 hours after the start of the test.
- All fish were observed as dead 3 hours after the start of the experiment at the two highest concentration levels of 50.0 and 100.0 mg/L (nominal).

BODY WEIGHT
- There was no considerable difference observed concerning body weights between the groups.

Reported statistics and error estimates:

The 24, 48, 72 and 96 hours LC50 values were calculated by Probit analysis using SPSS PC+ software.
The NOEC, LOEC and LC100 were determined directly from the raw data.

Sublethal observations / clinical signs:

Cumulative Mortality Data in the Definitive Test

Nominal concentration (mg/L)	Mortality [number of fish] (initial population = 7 fish)
	3 h	6 h	24 h	48 h	72 h	96 h
Control	0	0	0	0	0	0
6.25	0	0	0	0	0	0
12.5	0	0	0	0	2	2
25.0	0	0	7	-	-	-
50.0	7	-	-	-	-	-
100.0	7	-	-	-	-	-

 

Measured and Calculated Data of Bodyweight

Nominal Concentration (mg/L)	Measured Weight of 7 Fish (g)	Calculated Mean Weight of 1 fish (g)	Loading of Testing Aquarium (g fish/L testing Liquid)
Control	1.56	0.22	0.31
6.25	1.24	0.18	0.25
12.5	1.42	0.20	0.28
25.0	1.35	0.19	0.27
50.0	1.36	0.19	0.27
100.0	1.40	0.20	0.28

 

Analytical Results of the Test Samples

Test material (Nominal) (mg/L)	Renev. Cycle	Sampling	Test Material Meas. Average (mg/L)	Percentage of the Nominal Concentration	RSD (%)
Control	1	Start	ND	N/A	N/A
		End	ND	N/A	N/A
	4	Start	ND	N/A	N/A
		End	ND	N/A	N/A
6.25	1	Start	6.07	97.2	0.44
		End	5.73	91.6	0.25
	4	Start	6.10	97.6	0.21
		End	5.71	91.4	0.01
12.5	1	Start	12.6	100.4	0.31
		End	11.7	93.4	1.25
	4	Start	12.5	99.7	0.50
		End	12.3	98.8	0.13
25	1	Start	26.0	103.9	0.28
		End	25.8	103.2	0.18
50	1	Start	51.9	103.8	0.82
		End	51.9	103.8	0.83
100	1	Start	104.7	104.7	0.10
		End	104.7	104.7	0.35
	4	Start	104.8	104.8	0.31

 

Test Conditions: Temperature

Nominal Concentration (mg/L)	Water Temperature (°C)
	0 h*	24 h**	24 h*	48 h**	48 h*	72 h**	72 h*	96 h**
Control	21.8	20.7	21.8	20.6	22.2	20.9	22.2	20.5
6.25	21.8	20.7	21.9	20.6	22.2	20.9	22.3	20.5
12.5	21.9	20.8	21.9	20.6	22.3	21.0	22.3	20.6
25.0	22.0	20.8	-	-	-	-	-	-
50.0	22.2	20.8	-	-	-	-	-	-
100.0	22.3	20.8	-	-	-	-	-	-

* Measurement at the start of the renewal period in the fresh test medium.

** Measurement at the end of the renewal period in the old test medium.

 

Test Conditions: Dissolved Oxygen Conditions

Nominal Concentration (mg/L)	Water Temperature (°C)
	0 h*	24 h**	24 h*	48 h**	48 h*	72 h**	72 h*	96 h**
Control	99	88	97	86	99	82	99	81
6.25	99	84	97	82	99	80	98	81
12.5	99	83	97	83	99	80	99	80
25.0	99	89	-	-	-	-	-	-
50.0	99	91	-	-	-	-	-	-
100.0	99	89	-	-	-	-	-	-

* Measurement at the start of the renewal period in the fresh test medium.

** Measurement at the end of the renewal period in the old test medium.

 

Test Conditions: pH

Nominal Concentration (mg/L)	Water Temperature (°C)
	0 h*	24 h**	24 h*	48 h**	48 h*	72 h**	72 h*	96 h**
Control	6.32	6.59	6.34	6.31	6.30	6.30	6.44	6.33
6.25	6.41	6.70	6.48	6.46	6.77	6.32	6.49	6.47
12.5	6.54	6.78	6.56	6.47	6.86	6.42	6.61	6.52
25.0	6.83	6.81	-	-	-	-	-	-
50.0	6.81	6.88	-	-	-	-	-	-
100.0	6.82	6.97	-	-	-	-	-	-

* Measurement at the start of the renewal period in the fresh test medium.

** Measurement at the end of the renewal period in the old test medium.

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this acute fish toxicity study on Zebrafish (Brachydanio rerio) the observed and calculated endpoints for the effect of the test material were the following:
The 24h and 48h LC50 value: 17.85 mg/L (nominal) (95 % conf. limits: 12.01 – 24.75 mg/L)
The 72h and 96h LC50 value: 14.21 mg/L (nominal) (95 % conf. limits: 10.25 – 30.45 mg/L)
The 96h LC100 value: 25.0 mg/L (nominal)
The 96h No-Observed Effect Concentration (NOEC): 6.25 mg/L (nominal)
The 96h Lowest Observed Effect Concentration (LOEC): 12.5 mg/L (nominal)

Executive summary:

The acute toxicity of the test material was assessed with acute fish toxicity test on Zebrafish (Brachydanio rerio), over an exposure period of 96 hours in a semi-static system. The test was performed in accordance with the standardised guidelines OECD 203, EU Method C.1 and OCSPP 850.1075, under GLP conditions.

Due to problems with preparation of test material stock solution (opalescent test solution and precipitation when trying to dissolve it in aquarium water) deionised water was used as a test medium instead of aquarium water.

Because significant toxicity was observed at the highest examined concentration level during the preliminary range-finding test, five test concentrations in a geometric series with a separation factor of 2.0 and one untreated control were tested in the main experiment under semi-static conditions. The frequency of the water renewal periods was 24 hours.

The nominal concentrations of test material used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L. The test concentrations were analytically determined at the start and at the end of the first and at the last renewal periods. Concentration of the examined test groups of 25.0, 50.0 and 100.0 mg/L (nominal) was only analysed during the first renewal period, when all animals were observed as dead. The corresponding measured geometric mean test material concentrations were: 5.9; 12.3; 25.9; 51.9 and 104.7 mg/L. As the measured concentrations deviated not more than 20 per cent from the nominal in all cases, biological results are based on the nominal test material concentrations.

One aquarium was used in each test group and one in the control group. Each aquarium comprised seven fish and five litre test solution. The 24, 48, 72 and 96 hours LC50 values of the test material were calculated using Probit analysis by SPSS PC+ software. The NOEC and LOEC and LC100 values were determined from the raw data.

All achievable validity criteria were met during this study.

Under the conditions of this acute fish toxicity study on Zebrafish (Brachydanio rerio) the observed and calculated endpoints for the effect of the test material were the following:

The 24h and 48h LC50 value: 17.85 mg/L (nominal) (95 % conf. limits: 12.01 – 24.75 mg/L)

The 72h and 96h LC50 value: 14.21 mg/L (nominal) (95 % conf. limits: 10.25 – 30.45 mg/L)

The 96h LC100 value: 25.0 mg/L (nominal)

The 96h No-Observed Effect Concentration (NOEC): 6.25 mg/L (nominal)

The 96h Lowest Observed Effect Concentration (LOEC): 12.5 mg/L (nominal)

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

08 March 2018 to 12 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law) 1973

Version / remarks:

Amended 2009 under the reference of YAKUSHOKHATSU No. 1121002, SEIKYOKU No.2 and KANPOKIHATSU No. 021121002 and partially amended 2006 as the joint ordinance of The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 2-7-1-1 The guidelines related to the study reports for the registration application of pesticide

Version / remarks:

Ref. No. 12-Nousan-8147 on 24 November 2000) & Ref. No.13-Seisan-3986 on 10 October 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Analytical measurements were performed from the control and at the applied test concentration at the beginning and at the end of each renewal periods.
- The samples were analysed by an LC-MS method.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Because the test material is poorly soluble in water, a test solution was prepared using a saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.
- Saturated test material solution (100.0 mg/L nominal loading rate) was prepared by dispersing/dissolving the amount of test material into the test medium (aquarium water) two days before the start of the study. This solution was shaken for about 24 hours at approximately 30 °C and then equilibrated for about 24 hours at approximately 20 °C. The non-dissolved test material was removed by filtration through a fine (0.22 μm) filter to give appropriate 100 % saturated solution.
- As a Limit test was carried out, further dilution of stock solution was not performed.
- Since the test was performed under semi-static conditions, prior to the treatment, at each of the renewal periods, test material solution was prepared by the method described above.
- Test solutions renewals were performed at 24-hour intervals during the experiment.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Length at study initiation: 2.5 – 2.8 cm
- Weight at study initiation: Mean: 0.20-0.27 g

ACCLIMATION
- Acclimation period: More than 12 days
- Fish were bred in a well-known fish farm, under disease and parasite-controlled conditions. Fish were observed during the acclimatisation and test periods, the fish were healthy. There was no mortality of the population for seven days before the test.

FEEDING DURING TEST
- The fish were not fed during the test

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

189 mg/L (as CaCO3)

Test temperature:

20.3 and 21.6 °C

pH:

7.27 – 7.80

Dissolved oxygen:

71 – 100 % of the air saturation value

Nominal and measured concentrations:

Nominal: 100 % saturated solution. Biological results are based on the nominal test material concentration.
Geometric mean: 1.48 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Aquarium
- Material, size, headspace, fill volume: 5 L test solution
- Aeration: Test solutions were aerated during the test to avoid decreasing of dissolved oxygen concentration below the accepted level.
- Renewal rate of test solution: Test solutions renewals were performed at 24-hour intervals during the experiment.
- No. of organisms per vessel: 7
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- Biomass loading rate: Less than 1.0 g fish/litre testing liquid. Control: 0.37 g fish/L testing liquid and test material treated: 0.28 g fish/L testing liquid

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Aquarium water
- Intervals of water quality measurement: The water temperature, oxygen concentration and pH were measured at the start and at the end of the renewal periods during the experiment in each test aquarium.

OTHER TEST CONDITIONS
- Photoperiod: The light-dark cycle during the test was 16 hours light and 8 hours darkness.

EFFECT PARAMETERS MEASURED:
- The observations of fish were carried out in the following intervals: at 3, 6, 24, 48, 72 and 96 hours.
- The body weights of the fish were recorded before the introduction of fish. The loading of the aquaria was calculated on the basis of these body weights.

TEST CONCENTRATIONS
- Range finding study: A preliminary concentration range-finding test was conducted to determine the approximate toxicity of the test material so that appropriate test concentrations could be selected for use in the definitive test. Four fish were exposed to each concentration of the test material plus a control, for 96 hours under semi-static conditions (with 24-hour renewal in absence of stability data).
- Test concentrations: 0.1, 1, 10 and 100 % saturated solution.
- Results used to determine the conditions for the definitive study: Yes, because no toxic response was observed during the preliminary range-finding test, only one test concentration at the solubility level of the test material in the test medium (100.0 % saturated solution) and one control group was tested in the definitive study in a semi-static system.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 other: % saturated solution (1.48 mg/L, measured)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

VALIDITY
- The achievable validity criteria were within acceptable limits and therefore the study can be considered as valid.

CONCENTRATION OF THE TEST MATERIAL
- Test material concentration was analytically determined at the start and at the end of each renewal period.
- Measured concentration was below the Quantification Limit (LOQ) during the first renewal period and at the beginning of the second renewal period. In order to calculate the mean exposure concentrations, where the measured concentration was below the Quantification Limit, the concentration was taken as half of the Limit of Quantification (LOQ = 1.0 mg/L) (according to OECD 23; paragraph 3.3).
- Biological results are related to the nominal test material concentration.

MORTALITY DATA
- No mortality was observed in the control or test material group.
- No sublethal effect was observed.

BODY WEIGHT
- The body weight of 7 fish was weighed at the start of the test.
- There was no considerable difference observed concerning body weights between the groups.

Reported statistics and error estimates:

No statistical analysis was performed because of the lack of toxic effects.
The LC50, NOEC, LOEC and LC100 were determined directly from the raw data.

Sublethal observations / clinical signs:

Calculation of Exposure Concentrations

Nominal Concentration (% sat. sol.)	Measured Concentrations (mg/L)								Geometric Mean (mg/L)
	1st Renewal Period		2nd Renewal Period		3rd Renewal Period		4th Renewal Period
	Start	End	Start	End	Start	End	Start	End
Control	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	-
100	0.5*	0.5*	0.5*	3.29	2.49	2.92	2.84	2.68	1.48

nd: not detected

*: Concentration was below the Quantification Limit, in order to calculate mean value, concentration was taken as half of the Limit of Quantification (LOQ = 1.0 mg/L) according to OECD 23.

 

Cumulative Mortality Data in the Limit Test

Test Group	Cumulative Mortality (Number of Dead Fish) [Initial Population = 7 Fish]
	3 h	6 h	24 h	48 h	72 h	96 h
Control	0	0	0	0	0	0
100 % Saturated Solution	0	0	0	0	0	0

 

Measured and Calculated Data of Bodyweight

Test Group	Measured Weight of 7 Fish (g)	Calculated Mean Weight of 1 Fish (g)	Loading of Testing Aquarium (g Fish/L Testing Liquid)
Control	1.87	0.27	0.37
100 % Saturated Solution	1.41	0.20	0.28

 

Temperature

Test Group	Measuring
	0 h (1)	24 h (2)	24 h (1)	48 h (2)	48 h (1)	72 h (2)	72 h (1)	96 h (2)
Control	21.0	20.5	21.0	20.3	20.9	21.1	21.4	20.3
100 % Saturated Solution	21.6	20.5	21.5	20.3	21.4	21.3	21.0	20.3

(1): measurement at the start of the renewal period in the fresh test medium

(2): measurement at the end of the renewal period in the old test medium

 

Dissolved Oxygen

Test Group	Measuring
	0 h (1)	24 h (2)	24 h (1)	48 h (2)	48 h (1)	72 h (2)	72 h (1)	96 h (2)
Control	100	91	100	89	98	90	99	88
100 % Saturated Solution	71	88	73	92	72	93	71	92

(1): measurement at the start of the renewal period in the fresh test medium

(2): measurement at the end of the renewal period in the old test medium

 

pH

Test Group	Measuring
	0 h (1)	24 h (2)	24 h (1)	48 h (2)	48 h (1)	72 h (2)	72 h (1)	96 h (2)
Control	7.56	7.63	7.56	7.28	7.27	7.55	7.31	7.54
100 % Saturated Solution	7.62	7.73	7.67	7.41	7.45	7.61	7.80	7.53

(1): measurement at the start of the renewal period in the fresh test medium

(2): measurement at the end of the renewal period in the old test medium

 

Body Length

Test Group	Body Length (cm)
	1	2	3	4	5	6	7
Control	2.6	2.8	2.6	2.7	2.7	2.6	2.7
100 % Saturated Solution	2.7	2.6	2.5	2.6	2.8	2.6	2.6

 

RESULTS OF ANALYSIS

Measured Concentrations of the Second Renewal Period (24 h) with the 95 % Confidence Intervals

Nominal Conc. (% Saturated Solution)	Measured Concentrations at the Start	Measured Concentrations at the End
	mg/L	mg/L
Control	Not detected	Not detected
100	<LOQ	3.29 ± 1.863

 

Measured Concentrations of the Third Renewal Period (24 h) with the 95 % Confidence Intervals

Nominal Conc. (% Saturated Solution)	Measured Concentrations at the Start	Measured Concentrations at the End
	mg/L	mg/L
Control	Not detected	Not detected
100	2.49 ± 0.253	2.92 ± 1.016

 

Measured Concentrations of the Fourth Renewal Period (24 h) with the 95 % Confidence Intervals

Nominal Conc. (% Saturated Solution)	Measured Concentrations at the Start	Measured Concentrations at the End
	mg/L	mg/L
Control	Not detected	Not detected
100	2.84 ± 0.117	2.68 ± 1.066

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the test material had no toxic effect at aquatic saturation (1.48 mg/L, measured) on fish; the LC50 results and the LOEC are higher than the solubility level of the test material in the test medium.

Executive summary:

The acute toxicity of the test material to fish was investigated in accordance with the standardised guidelines OECD 203, EU Method C.1., EPA OCSPP 850.1075 and Japanese guidelines, under GLP conditions.

The acute toxicity of the test material was assessed with acute fish toxicity test on Zebrafish (Brachydanio rerio), over an exposure period of 96 hours in a semi-static system.

Because no toxic response was observed during the preliminary range-finding test, only a Limit test was carried out using only one test concentration at the solubility level of the test material in the test medium (100.0 % saturated solution) and one control group in the definitive study under semi-static conditions.

The test concentration was analytically determined at the start and at the end of the renewal periods. The measured geometric mean test material concentration was 1.48 mg/L. The biological results are based on the nominal test material concentration.

One aquarium was used in the test group and one in the control group. Each aquarium comprised seven fish and five litre test solution.

All achievable validity criteria were met during this study.

Under the conditions of this study, the test material had no toxic effect at aquatic saturation (1.48 mg/L, measured) on fish; the LC50 results and the LOEC are higher than the solubility level of the test material in the test medium.

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

See the read-across report attached in Section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

48 h

Dose descriptor:

EC50

Remarks:

and 24 h

Effect conc.:

17.85 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: (95 % conf. limits: 12.01 - 24.75 mg/ L)

Key result

Duration:

96 h

Dose descriptor:

LC50

Remarks:

and 72 h

Effect conc.:

14.21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: (95 % conf. limits: 10.25 – 30.45 mg/L)

Key result

Duration:

96 d

Dose descriptor:

LC100

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Reference 4

Endpoint:

short-term toxicity to fish

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

See the read-across report attached in Section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 other: % saturated solution (1.48 mg/L, measured)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Reference 5

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

27 May 2013 to 31 May 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with GLP and agreed protocols, but it was not possible to derive a reliable analytical method to confirm the content of the test solutions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

Test solutions were prepared in accordance with the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23

Deviations:

yes

Remarks:

- due to the nature of the substance, a suitable analytical method could not be derived to adequately confirm the concentration of the test material in solution

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

yes

Remarks:

- due to the nature of the substance, a suitable analytical method could not be derived to adequately confirm the concentration of the test material in solution

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Deviations:

yes

Remarks:

- due to the nature of the substance, a suitable analytical method could not be derived to adequately confirm the concentration of the test material in solution

Qualifier:

according to guideline

Guideline:

other: JMAFF 2-7-1-1 The guidelines related to the study reports for the registration application of pesticide (Ref. No. 12-Nousan-8147 on 24 November 2000) & Ref. No.13-Seisan-3986 on 10 October 2001.

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Chemical Substance Control Law 1973, amended 2009 under the reference of YAKUSHOKHATSU No. 1121002, SEIKYOKU No.2 and KANPOKIHATSU No. 021121002 and partially amended 2006 as the joint ordinance of Japanese METI, MHLW and MOE guidance for studies.

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A supersaturated test material solution (nominal loading rate of 100 mg/L) was prepared by dispersing/dissolving the appropriate amount of test material into the test medium (aquarium water) two days before the start of the experiment. This solution was shaken for about 24 hours at approximately 30 °C and then equilibrated for around 24 hours at the test temperature. Undissolved test material was removed by filtration through a fine (0.22 μm) filter to give a 100 % v/v saturated solution.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Length at study initiation: 2.7 to 3.3 cm
- Weight at study initiation: Average of 0.296 g for the control and 0.303 g for the test group
- Method of breeding: Fish were bred in a well-known fish farm, under disease- and parasite-controlled conditions.
- Feeding during test: Fish were not fed during the test

ACCLIMATION
- Acclimation period: At least 12 days
- Health during acclimation (any mortality observed): Mortality of less than 5 per cent of the population was recorded for seven days before the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

54 mg/L as CaCO₃

Test temperature:

21.0 to 21.9 °C

pH:

8.00 to 8.47

Dissolved oxygen:

62 to 99 % of the air saturation value

Nominal and measured concentrations:

- Nominal concentration: 100 % (v/v) saturated solution prepared as a loading rate of 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Aquaria
- Fill volume: 5 L
- Renewal rate of test solution (frequency): Every 24 hours
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: < 1.0 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Aquarium water

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Mortality and any sub-lethal effect were determined at 3, 6, 24, 48, 72 and 96 hours. The test conditions (pH, temperature, oxygen saturation) were examined at the start and at the end of each renewal period.

TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: 0.1, 1, 10 and 100 % (v/v) saturated solutions
- Results used to determine the conditions for the definitive study: Yes. Three animals were used in each group. No mortality was seen in any group. Due to the lack of a toxic response, the definitive test was therefore performed as a limit test.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: None observed
- Observations on body weight: There was no considerable difference observed concerning body weight between the control and treated group
- Mortality of control: 0 % mortality occurred in the control group
- Mortality of treatment group: 0 % mortality was observed

Reported statistics and error estimates:

No statistical analysis was performed because of the lack of toxic effects. All values were concluded directly from the raw data.

Sublethal observations / clinical signs:

Table 1: Cumulative mortality

Test Group	Mortality Rate (Dead Fish / Treated Fish)
	3 hours	6 hours	24 hours	48 hours	72 hours	96 hours
Control	0/7	0/7	0/7	0/7	0/7	0/7
100 % (v/v) saturated solution	0/7	0/7	0/7	0/7	0/7	0/7

 

Table 2: Test conditions

Test Group		Water Quality Measurement Intervals
		0 hours	24 hours		48 hours		72 hours		96 hours
	Renewal Period	Start#	End*	Start	End	Start	End	Start	End
Control	Dissolved oxygen (% saturation)	88	96	98	97	99	96	97	96
100 % (v/v) saturated solution		62	98	63	98	62	98	63	95
Control	pH	8.23	8.41	8.21	8.43	8.47	8.42	8.35	8.32
100 % (v/v) saturated solution		8.00	8.21	8.11	8.30	8.10	8.28	8.13	8.22
Control	Temperature (°C)	21.7	21.8	21.6	21.8	21.5	21.7	21.9	21.6
100 % (v/v) saturated solution		21.0	21.4	21.2	21.7	21.6	21.7	21.7	21.6

# New media at the start of the renewal period

* Old media at the end of the 24 hour renewal period

Validity criteria fulfilled:

no

Remarks:

All validity criteria were fulfilled apart from those relating to test concentration maintenance. A suitable analytical method could not be derived to confirm the test concentrations.

Conclusions:

Under the conditions of the test, no mortality was observed. The LOEC, LC50 and LC100 were all found to be greater than 100 % (v/v) saturated solution (100 mg/L nominal loading rate). The NOEC was determined to be 100 % (v/v) saturated solution (100 mg/L nominal loading rate).

Executive summary:

The acute toxicity of the test material was determined in Zebrafish under semi-static conditions for 96 hours in accordance with the standardised guidelines OECD 203, EU Method C.3, EPA OPPTS 850.1075, JMAFF 2-7-1-1 and the Chemical Substance Control Law 1973. Due to the nature of the test material, the test concentrations for the study were prepared in accordance with the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23. The study was performed under GLP conditions.

An initial range-finding test was performed which demonstrated no toxicity up to the highest concentration tested. Therefore a Limit test was performed for the highest achievable concentration in the definitive test. The test material was tested as a 100 % v/v saturated solution, prepared using a loading rate of 100 mg/L.

Seven fish were exposed to the 100 % (v/v) saturated solution for 96 hours. The test media was renewed at 24 hour intervals. Mortality and any sub-lethal effect were determined at 3, 6, 24, 48, 72 and 96 hours.

No mortality was seen in either the control group or the treated group. No abnormal behaviour was reported and there was no considerable difference observed concerning body weight between the control and treated group.

As a suitable analytical method could not be derived for this study, it was not possible to fulfil all the validity criteria stated by the quoted guidelines; however, all other validity criteria not dependent on confirming the maintenance of the test concentration were satisfactorily completed.

Under the conditions of the test, no mortality was observed. The LOEC, LC50 and LC100 were all found to be greater than 100 % (v/v) saturated solution (100 mg/L nominal loading rate). The NOEC was determined to be 100 % (v/v) saturated solution (100 mg/L nominal loading rate).

Description of key information

Key Study on Read Across Substance HS-21P-T: Sipos (2018)
Under the conditions of this acute fish toxicity study on Zebrafish (Brachydanio rerio) the observed and calculated endpoints for the effect of the test material were the following:
The 24h and 48h LC50 value: 17.85 mg/L (nominal) (95 % conf. limits: 12.01 – 24.75 mg/L)
The 72h and 96h LC50 value: 14.21 mg/L (nominal) (95 % conf. limits: 10.25 – 30.45 mg/L)
The 96h LC100 value: 25.0 mg/L (nominal)
The 96h No-Observed Effect Concentration (NOEC): 6.25 mg/L (nominal)
The 96h Lowest Observed Effect Concentration (LOEC): 12.5 mg/L (nominal)

Supporting Study on Target Substance: Hajba-Horváth (2013)
96 hour LOEC, LC50 and LC100 > 100 % (v/v) saturated solution (100 mg/L nominal loading rate) and 96 hour NOEC 100 % (v/v) saturated solution (100 mg/L nominal loading rate), OECD 203, EU Method C.3, EPA OPPTS 850.1075, JMAFF 2-7-1-1 and the Chemical Substance Control Law 1973.

Supporting Study on Read Across Substance GS-11P-T: Sipos (2018)
Under the conditions of this study, the test material had no toxic effect at aquatic saturation (1.48 mg/L, measured) on fish; the LC50 results and the LOEC are higher than the solubility level of the test material in the test medium.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

14.21 mg/L

Additional information

Supporting Study on Target Substance: Hajba-Horváth (2013)

In the supporting study Hajba-Horváth (2013), the acute toxicity of the test material was determined in Zebrafish under semi-static conditions for 96 hours in accordance with the standardised guidelines OECD 203, EU Method C.3, EPA OPPTS 850.1075, JMAFF 2-7-1-1 and the Chemical Substance Control Law 1973. Due to the nature of the test material, the test concentrations for the study were prepared in accordance with the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23. The study was performed under GLP conditions. The study was therefore assigned a reliability score of 1 in accordance with the principles for assessing data quality as defined in Klimisch et al. (1997).

An initial range-finding test was performed which demonstrated no toxicity up to the highest concentration tested. Therefore a Limit test was performed for the highest achievable concentration in the definitive test. The test material was tested as a 100 % v/v saturated solution, prepared using a loading rate of 100 mg/L.

Seven fish were exposed to the 100 % (v/v) saturated solution for 96 hours. The test media was renewed at 24 hour intervals. Mortality and any sub-lethal effect were determined at 3, 6, 24, 48, 72 and 96 hours.

No mortality was seen in either the control group or the treated group. No abnormal behaviour was reported and there was no considerable difference observed concerning body weight between the control and treated group.

As a suitable analytical method could not be derived for this study, it was not possible to fulfil all the validity criteria stated by the quoted guidelines; however, all other validity criteria not dependent on confirming the maintenance of the test concentration were satisfactorily completed.

Under the conditions of the test, no mortality was observed. The LOEC, LC50 and LC100 were all found to be greater than 100 % (v/v) saturated solution (100 mg/L nominal loading rate). The NOEC was determined to be 100 % (v/v) saturated solution (100 mg/L nominal loading rate).

 

Key Study on Read Across Substance HS-21P-T: Sipos (2018)

In the key study on target substance HS-21P-T Sipos (2018), the acute toxicity of the test material was assessed with acute fish toxicity test on Zebrafish (Brachydanio rerio), over an exposure period of 96 hours in a semi-static system. The test was performed in accordance with the standardised guidelines OECD 203, EU Method C.1 and OCSPP 850.1075, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Due to problems with preparation of test material stock solution (opalescent test solution and precipitation when trying to dissolve it in aquarium water) deionised water was used as a test medium instead of aquarium water.

Because significant toxicity was observed at the highest examined concentration level during the preliminary range-finding test, five test concentrations in a geometric series with a separation factor of 2.0 and one untreated control were tested in the main experiment under semi-static conditions. The frequency of the water renewal periods was 24 hours.

The nominal concentrations of test material used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L. The test concentrations were analytically determined at the start and at the end of the first and at the last renewal periods. Concentration of the examined test groups of 25.0, 50.0 and 100.0 mg/L (nominal) was only analysed during the first renewal period, when all animals were observed as dead. The corresponding measured geometric mean test material concentrations were: 5.9; 12.3; 25.9; 51.9 and 104.7 mg/L. As the measured concentrations deviated not more than 20 per cent from the nominal in all cases, biological results are based on the nominal test material concentrations.

One aquarium was used in each test group and one in the control group. Each aquarium comprised seven fish and five litre test solution. The 24, 48, 72 and 96 hours LC50 values of the test material were calculated using Probit analysis by SPSS PC+ software. The NOEC and LOEC and LC100 values were determined from the raw data.

All achievable validity criteria were met during this study.

Under the conditions of this acute fish toxicity study on Zebrafish (Brachydanio rerio) the observed and calculated endpoints for the effect of the test material were the following:

The 24h and 48h LC50 value: 17.85 mg/L (nominal) (95 % conf. limits: 12.01 – 24.75 mg/L)

The 72h and 96h LC50 value: 14.21 mg/L (nominal) (95 % conf. limits: 10.25 – 30.45 mg/L)

The 96h LC100 value: 25.0 mg/L (nominal)

The 96h No-Observed Effect Concentration (NOEC): 6.25 mg/L (nominal)

The 96h Lowest Observed Effect Concentration (LOEC): 12.5 mg/L (nominal)

 

Supporting Study on Read Across Substance GS-11P-T: Sipos (2018)

In the supporting study on target substance GS-11P-T Sipos (2018), the acute toxicity of the test material to fish was investigated in accordance with the standardised guidelines OECD 203, EU Method C.1., EPA OCSPP 850.1075 and Japanese guidelines, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The acute toxicity of the test material was assessed with acute fish toxicity test on Zebrafish (Brachydanio rerio), over an exposure period of 96 hours in a semi-static system.

Because no toxic response was observed during the preliminary range-finding test, only a Limit test was carried out using only one test concentration at the solubility level of the test material in the test medium (100.0 % saturated solution) and one control group in the definitive study under semi-static conditions.

The test concentration was analytically determined at the start and at the end of the renewal periods. The measured geometric mean test material concentration was 1.48 mg/L. The biological results are based on the nominal test material concentration.

One aquarium was used in the test group and one in the control group. Each aquarium comprised seven fish and five litre test solution.

All achievable validity criteria were met during this study.

Under the conditions of this study, the test material had no toxic effect at aquatic saturation (1.48 mg/L, measured) on fish; the LC50 results and the LOEC are higher than the solubility level of the test material in the test medium.