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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

22 March 2017 to 24 March 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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: YAKUSHOKHATSU No. 1121002, SEIKYOKU No.2 and KANPOKIHATSU No. 021121002

Version / remarks:

The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE). 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 2-7-2-1

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:

- The test concentrations were analytically determined at the start and at the end of the first and at the last renewal periods.
- Analytical measurements were performed from the control and at the applied test concentration levels at the beginning and at the end of the renewal periods.
- The samples were analysed by a LC-MS method.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Even if the test item seems to be soluble in ISO medium, but strong precipitation is observed within minutes, it is analytically confirmed that test item does not present in the solution anymore. For this reason the experiment was carried out in a different media, in which we could keep the test item in solution.
- Due to problems with preparation of test material stock solution (strong precipitation) 1:1 v/v solution of ISO medium and deionised water was used as a test medium instead of ISO medium.
- A saturated test material solution (nominal loading rate of 100 mg/L) was prepared with direct addition of the test material, mixed into the test medium (1:1 v/v solution of ISO medium and deionised water) using ultrasonic bath (10 minutes). Saturated test material solution was opalescent. The test solutions were prepared by appropriate dilution of this stock solution just before introduction of the Daphnia (start of the treatments).
- Prior to the treatment, at each of the renewal periods test solutions were prepared by the method described above.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Age: Less than 24 h old at the beginning of the test.
- Feeding during test: No

ACCLIMATION
- Acclimation period: There was no acclimatisation because the water used was similar to the culture water.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

The reconstituted water (ISO medium) had a total hardness of 247 mg/L (as CaCO3). The dilution water (1:1 v/v solution of ISO medium and deionised water) used in the experiment had a total hardness of 118 and 121 mg/L (as CaCO3), respectively at the start of the treatments.

Test temperature:

20.6 to 21.0 °C

pH:

6.93 to 7.59.

Dissolved oxygen:

8.0 to 9.1 mg/L

Nominal and measured concentrations:

- Nominal concentrations: 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solutions.
- Measured geometric mean test material concentrations were: 3.56, 5.48, 11.19, 22.64 and 39.60 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers
- Renewal rate of test solution: The frequency of the water renewal periods was 24 hours due to precipitation observed after 24 hours during the preliminary range-finding test.
- Material, size, headspace, fill volume: Glass beakers with at least 5 mL test solution / animal.
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per vehicle control: 4

TEST MEDIUM / WATER PARAMETERS
- Reconstituted water (1:1 v/v solution of ISO medium and deionised water) was used as dilution water for both the range finding and definitive tests.
- Reconstituted water (ISO medium) was used for breeding the test animals.
- Composition of ISO medium: Separate stock solutions of individual trace elements were first prepared in deionised water at the concentrations below. The medium was prepared of these different stock solutions, so it contained all trace elements (combined solution). 25 mL/L of each stock solution was added to deionised water to prepare the media.
- CaCl2.2H2O: 11.76 g/L
- MgSO4.7H2O: 4.93 g/L
- KCl: 0.23 g/L
- NaHCO3: 2.59 g/L
- Particulate matter: Checked and guideline compliant.
- Total organic carbon: Checked and guideline compliant.


OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test. The pH was measured at
the start and at the end of the renewal periods in each test vessel and was in the range of 6.93 – 7.63.
- Photoperiod: The test was carried out in 16-hour light and 8-hour dark cycle.
- Temperature: The water temperature was measured at the start and at the end of the renewal in each test vessel. The test temperature was in
the range of 20.6 – 21.0°C measured in the test vessels. The additionally measured temperature in the climate chamber was between 20.5 and 21.2°C.
- Oxygen concentration: The dissolved oxygen concentration was measured in each test vessel at the start and at the end of the renewal periods and was in the range of 8.0 – 9.1 mg/L.

EFFECT PARAMETERS MEASURED:
- The immobility or mortality of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile.
- The number of immobilised animals and the percentage of immobility were determined at 24 and 48 hours.
- The water temperature, the oxygen concentrations and pH of the controls and the test material solution were measured at the beginning and at the end of the renewal periods.

VEHICLE CONTROL PERFORMED: Yes

RANGE-FINDING STUDY
- A 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. Ten daphnids (divided into two replicates) in each test concentration and control were exposed for 48 hours under semi-static conditions.
- Test concentrations: 0.1, 1, 10 and 100 % saturated solutions preparaed by appropriate dilution of the stock solutions.
- Results used to determine the conditions for the definitive study: Yes. At 100 % saturated solution, all of the daphnia were immobilised in the preliminary test.
- Due to precipitation observed after 24 hours during the preliminary range-finding test the experiment was performed under semi-static conditions.
- Because significant immobility was observed during the preliminary concentration range-finding test, five test concentrations in a geometric series (factor 2.0) and one control group were tested in the main experiment under semi-static conditions.
- The biological results are based on the measured geometric mean test material concentrations

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 39.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

26.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: (95 % CI: 22.41 to 31.57 mg/L)

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 39.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

22.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

39.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

CONCENTRATIONS OF THE TEST MATERIAL
- Test concentrations were analytically determined at the beginning and at the end of the renewal periods. Samples from the control were analysed at the start and at the end of the renewal periods as well.
- The following nominal concentrations were tested: 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solution.
- The corresponding measured geometric mean test item concentrations were: 3.56, 5.48, 11.19, 22.64 and 39.60 mg/L.
- The biological results are based on the measured geometric mean concentrations.

IMMOBILISATION
- The number of immobilised animals and the percentage of immobility were determined at the 24th and 48th hour (Table 1). In addition to immobility, no abnormal behaviour or appearance of test animals was detected.
- Although 10 % immobilisation could be observed at the test item groups of 6.25, 12.5, 25.0 and 50.0 % saturated solution, these differences were not statistically
significant compared to the control and are negligible due to that the accepted control immobilisation is 10 %.

Results with reference substance (positive control):

- For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
- The date of the last study (Study Code: 17/058-023DA) with reference material Potassium dichromate (batch no.: A0345704) is: 08 - 09 March 2017.
- The 24h EC50: 0.64 mg/L, (95 % confidence limits: 0.59 – 0.68 mg/L)

Reported statistics and error estimates:

- The 24 hours EC50 values of the test material could not be calculated due to the slight effects observed. The 48 hours EC50 values were calculated using Probit analysis by TOXSTAT software. The 24 h EC50 and 48 h EC100 values of the test material were determined directly from the raw data.
- For the determination of the LOEC and NOEC, the immobilisation at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software.

Number and percentage of immobilised animals

Concentration (mg/L)		Number of animals	Immobilised animals
			24 hours		48 hours
Nominal	Measured		number	percent	number	percent
Control	0.0	20	0	0	0	0
6.25	3.56	20	2	10	2	10
12.5	5.48	20	0	0	2	10
25.0	11.19	20	1	5	2	10
50.0	22.64	20	0	0	2	10
100.0	39.60	20	2	10	19	95*

* statistically significantly different compared to the control values (Dunnett’s Test; α = 0.05)

 

Results of the Preliminary Range-Finding Study

Nominal Concentrations [% Sat. Solution]	Untreated Control (ISO)	Untreated Control (50 % ISO)	0.1	1	10	100
Number of treated/ immobilised animals	10/0	10/0	10/0	10/0	10/0	10/0

 

Calculation of Exposure Concentrations

Nominal Concentrations (% Sat. Sol.)	Measured Concentrations (mg/L) 1^st Renewal Period		Measured Concentrations (mg/L) 2nd Renewal Period		Geometric Mean (mg/L)
	Start	End	Start	End
Control	Not detected	Not detected	Not detected	Not detected	-
6.25	2.82	3.77	3.33	4.55	3.56
12.5	5.03	5.55	5.19	6.22	5.48
25.0	10.5	11.3	10.4	12.7	11.19
50.0	22.5	21.2	22.4	24.6	22.64
100.0	42.7	34.6	39.3	41.4	36.60

 

Temperature Measured in the Test Vessels (°C)

Concentration		Replicate	Measuring Time
Nominal (% Sat. Sol.)	Measured (mg/L)		0 h (Fresh Media)	24 h (Old Media)	24 h (Fresh Media)	48 h (Old Media)
Control	0.0	1	20.7	20.6	20.9	20.7
		2	20.7	20.6	20.9	20.7
		3	20.7	20.6	20.9	20.7
		4	20.7	20.6	20.9	20.7
6.25	3.56	1	20.7	20.6	20.9	20.7
		2	20.7	20.6	20.9	20.7
		3	20.7	20.6	20.9	20.7
		4	20.7	20.6	20.9	20.7
12.5	5.48	1	20.7	20.6	20.9	20.7
		2	20.7	20.6	20.9	20.7
		3	20.7	20.6	20.9	20.7
		4	20.7	20.6	20.9	20.7
25.0	11.19	1	20.7	20.6	20.9	20.7
		2	20.7	20.6	20.9	20.7
		3	20.7	20.6	20.9	20.7
		4	20.7	20.6	20.9	20.7
50.0	22.64	1	20.7	20.7	20.9	20.7
		2	20.7	20.7	20.9	20.7
		3	20.7	20.7	20.9	20.7
		4	20.7	20.7	20.9	20.7
100.0	39.60	1	20.7	20.7	20.9	20.7
		2	20.7	20.7	21.0	20.7
		3	20.7	20.7	21.0	20.7
		4	20.7	20.7	21.0	20.7

 

Oxygen Concentration Measured in the Test Vessels (mg/L)

Concentration		Replicate	Measuring Time
Nominal (% Sat. Sol.)	Measured (mg/L)		0 h (Fresh Media)	24 h (Old Media)	24 h (Fresh Media)	48 h (Old Media)
Control	0.0	1	8.8	8.6	8.7	9.1
		2	8.8	8.6	8.7	9.0
		3	8.8	8.6	8.7	9.1
		4	8.8	8.6	8.7	9.1
6.25	3.56	1	8.7	8.4	8.5	8.4
		2	8.7	8.4	8.5	8.4
		3	8.7	8.3	8.5	8.6
		4	8.7	8.4	8.5	8.2
12.5	5.48	1	8.7	8.4	8.5	8.2
		2	8.7	8.4	8.5	8.4
		3	8.7	8.4	8.5	8.4
		4	8.7	8.3	8.5	8.5
25.0	11.19	1	8.6	8.3	8.5	8.5
		2	8.6	8.3	8.5	8.3
		3	8.6	8.3	8.5	85
		4	8.6	8.3	8.5	8.4
50.0	22.64	1	8.6	8.2	8.5	8.5
		2	8.6	8.1	8.5	8.1
		3	8.6	8.1	8.5	8.0
		4	8.6	8.2	8.5	8.2
100.0	39.60	1	8.5	8.2	8.4	8.2
		2	8.5	8.1	8.4	8.1
		3	8.5	8.1	8.4	8.2
		4	8.5	8.1	8.4	8.2

 

pH Measured in the Test Vessels

Concentration		Replicate	Measuring Time
Nominal (% Sat. Sol.)	Measured (mg/L)		0 h (Fresh Media)	24 h (Old Media)	24 h (Fresh Media)	48 h (Old Media)
Control	0.0	1	7.16	7.41	6.93	7.45
		2	7.16	7.55	6.93	7.48
		3	7.16	7.59	6.93	7.50
		4	7.16	7.63	6.93	7.52
6.25	3.56	1	7.28	7.58	7.08	7.55
		2	7.28	7.57	7.08	7.57
		3	7.28	7.57	7.08	7.55
		4	7.28	7.57	7.08	7.55
12.5	5.48	1	7.29	7.54	7.11	7.54
		2	7.29	7.54	7.11	7.54
		3	7.29	7.53	7.11	7.55
		4	7.29	7.54	7.11	7.55
25.0	11.19	1	7.29	7.53	7.11	7.55
		2	7.29	7.51	7.11	7.52
		3	7.29	7.52	7.11	7.52
		4	7.29	7.51	7.11	7.52
50.0	22.64	1	7.25	7.46	7.10	7.50
		2	7.25	7.44	7.10	7.47
		3	7.25	7.44	7.10	7.46
		4	7.25	7.43	7.10	7.44
100.0	39.60	1	7.22	7.30	7.08	7.22
		2	7.22	7.26	7.08	7.19
		3	7.22	7.24	7.08	7.17
		4	7.22	7.22	7.08	7.16

 

 

Validity

- There was no immobilisation in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L.

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

 

Analytical Determinations

Calibration

Summary of Standard Curve Parameters

Analytical Occasion	C	B	A^2	Correlation Coefficient
22 March 2017	-3.44 x 10^5	3.13 x 10^6	7.59 x 10^3	0.999
23 March 2017	9.55 x 10^5	3.67 x 10^6	9.40 x 10^3	0.99
24 March 2017	-1.90 x 10^6	3.05 x 10^6	8.62 x 10^3	0.997

 

Results of the Analysis

Measured Concentrations with the 95 % Confidence Intervals

Concentration, % of Sat. Sol.	Measured Concentrations at the First Renewal Period (mg/L)
	Start	End
Control	Not detected	Not detected
6.25	2.82 ± 0.056	3.77 ± 0.638
12.5	5.03 ± 0.357	5.55 ± 0.218
25	10.5 ± 0.70	11.3 ± 1.24
50	22.5 ± 0.82	21.2 ± 1.52
100	43.7 ± 0.78	34.6 ± 2.96

 

Measured Concentrations with the 95 % Confidence Intervals

Concentration, % of Sat. Sol.	Measured Concentrations at the Second Renewal Period (mg/L)
	Start	End
Control	Not detected	Not detected
6.25	3.33 ± 0.528	4.55 ± 0.601
12.5	5.19 ± 0.420	6.22 ± 0.492
25	10.4 ± 0.88	12.7 ± 1.79
50	22.4 ± 0.74	24.6 ± 2.19
100	39.3 ± 0.94	41.4 ± 4.04

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the 48-h EC50 was 26.60 mg/L (measured) with 95 % CI of 22.41 to 31.57 mg/L. The 48-h EC100 was > 39.60 mg/L, the 48-h NOEC was 22.64 mg/L and the 48-h LOEC was 39.60 mg/L.

Executive summary:

The acute toxicity of the test material was investigated in accordance with the standardised guidelines OECD 202, EU Method C.2, EPA OCSPP 850.1010 and Japanese guidelines under GLP conditions. Testing was performed on Daphnia magna in an acute immobilisation test, over an exposure period of 48 hours in a semi-static test system.

Even though the test material seems to be soluble in the test media, strong precipitation was observed within minutes. For this reason the experiment was carried out in a different media. A 1:1 v/v solution of ISO medium and deionised water was used as a test medium instead of ISO medium.

Because significant immobility 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 test under semi-static conditions.

The nominal concentrations of test material used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solution.

The test concentrations were analytically determined at the start and at the end of the renewal periods. The corresponding measured geometric mean test material concentrations were: 3.56; 5.48; 11.19; 22.64 and 39.60 mg/L. Biological results are based on the measured geometric mean test material concentrations.

Twenty animals, divided into four groups of five animals each were used at the test concentrations and for the control as well.

The 24 hours EC50 values of the test material could not be calculated due to the slight effects observed. The 48 hours EC50 values were calculated using Probit analysis by TOXSTAT software. The 24 h EC50 and 48 h EC100 values of the test material were determined directly from the raw data. For the determination of the LOEC and NOEC, the immobilisation at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software.

All validity criteria were met during this study.

Under the conditions of the study the 48-h EC50 was 26.60 mg/L with 95 % CI of 22.41 to 31.57 mg/L. The 48-h EC100 was > 39.60 mg/L, the 48-h NOEC was 22.64 mg/L and the 48-h LOEC was 39.60 mg/L.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 August 2008 to 29 August 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with GLP and agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Signature: 15/10/2007; Date of Inspection: 21/08/2007

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.7 mg/L test concentration
- Method: Water samples were taken from the control (replicates R1– R4 pooled) and the test group (replicates R1– R2 and R3– R4 pooled) at 0 and 48 hours for quantitative analysis. Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.
- Sample storage conditions before analysis: Not applicable, samples were analysed immediately after being taken.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Controls:
A positive control, conducted approximately every six months, used potassium dichromate as the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.

Media preparation trial:
Pre-study solubility work conducted indicated that, whilst the test material was readily soluble in deionised reverse osmosis water, upon addition to reconstituted water the test material was observed to form a dispersion.
Based on this information the test material was categorised as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.
An amount of test material (1100 mg) was dispersed in 11 litres of reconstituted water with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21 °C for 24 hours. After the stirring period samples were taken for chemical analysis after the following pre-treatments:
> Filtration through a 0.2 µm Sartorius Sartopore filter (initial 1 litre discarded in order to pre-condition the filter)
> Filtration through a 0.2 µm Sartorius Sartopore filter (initial 2 litres discarded in order to pre-condition the filter)

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: 1st instar Daphnia magna
- Source: Derived from in-house laboratory cultures
- Age at study initiation (mean and range, SD): Less than 24 hours old
- Method of breeding: Parthenogenesis.
- Feeding during test
- Food type: Suspension of algae (Chlorella sp.)
- Frequency: Daily

ACCLIMATION
- Acclimation period: 24 hours
- Acclimation conditions: Temperature controlled at 21 °C
- Type and amount of food: Each culture was fed daily with a suspension of algae (Chlorella sp.)
- Feeding frequency: Daily

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

Theoretical total hardness of 250mg/L as CaCO3

Test temperature:

The temperature was maintained at 22°C. The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

pH:

At 0 hours: pH = 7.9; pH of the Test material
At 48 hours: pH = 8.0 = control
The pH was recorded at the start and termination of the test using a WTW pH/Oxi 340I pH

Dissolved oxygen:

Dissolved oxygen concentrations were recorded at the start and termination of the test using an oxygen meter.

Nominal and measured concentrations:

Nominal concentration in definitive test: 1.7 mg/L
Nominal concentrations in range finding test: 0.17 and 1.7 mg/L

Measured: Verification of test concentrations - Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be less than the limit of quantitation (LOQ) of the analytical method.
This does not infer that no test material was in solution but that the dissolved concentration (i.e. bioavailable to the test organisms) was below the limit of quantitation which was assessed down to 0.50 mg/L.
The measured test concentration gave an estimate of the dissolved concentration of the test material and hence bioavailable to the test organisms.
During the media preparation trial a test concentration of 1.7 mg/L was attained; however during the definitive test the test material could not be determined in the test media. This was considered to be due to slight differences in stirring speeds and/or slight differences in water quality.
Based on this information the results are based on concentration as % v/v saturated solution.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass jars.
- Type: closed
- Material, size, headspace, fill volume: 250 mL glass jars containing approximately 200 mL of test preparation were used.
- Aeration: No aeration
- No. of organisms per vessel: 5 daphnids
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water
i) Stock Solutions:
a) CaCl2.2H2O 11.76 g/L
b) MgSO4.7H2O 4.93 g/L
c) NaHCO3 2.59 g/L
d) KCl 0.23 g/L

ii) Preparation:
An aliquot (25 mL) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm^-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.

OTHER TEST CONDITIONS
- Adjustment of pH: No adjustment during test period.
- Photoperiod: 16 hours light and 8 hours darkness with 20 minutes dawn and dusk transitions periods.

EFFECT PARAMETERS MEASURED
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.17 and 1.7 mg/L
- Results used to determine the conditions for the definitive study: Yes, the test concentration to be used in the definitive test was determined by a preliminary range-finding test.

Reference substance (positive control):

yes

Remarks:

A positive control conducted approximately every six months used potassium dichromate as the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 other: % v/v

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Results: No immobilisation was observed at the nominal test concentrations of 0.17 and 1.7 mg/L.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test media preparations were observed to be clear, colourless solutions throughout the duration of the test.

Results with reference substance (positive control):

The No Observed Effect Concentration after 24 and 48 hours were 0.56 and 0.32 mg/L respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
- EC50/LC50: 0.70mg/L (with 95% confidence limits of 0.62 – 0.80 mg/L)

Reported statistics and error estimates:

The slope and standard error of the response curve at 48 hours was 11 (SE = 2.3). Due to the unsuitable nature of the data it was not possible to calculate the slope and error of the response curve at 24 hours.
The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.78 mg/L (sd = 0.22).

Media preparation trial

The results of the media preparation trial showed measured test concentrations from the saturated solution method of preparation to be approximately 1.7 mg/L following filtration through a 0.2 µm filter with the first 1 or 2 litres discarded. 

Based on the results of the media preparation trial it was considered that the use of a saturated solution was the most appropriate method of preparation. The test material was prepared by stirring 100 mg/L using a propeller stirrer at approximately 1500 rpm for 24 hours. The undissolved test material was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres discarded) to give a saturated solution of the test material.

Range-finding Test

Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the range-finding test are given in Table 1.

No immobilisation was observed at the nominal test concentrations of 0.17 and 1.7 mg/L. 

Based on this information, a single nominal test concentration of four replicates, of 1.7 mg/L was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the highest attainable test concentration of 1.7 mg/L, no immobilisation or adverse reactions to exposure were observed.

DefinitiveTest

Verification of test concentrations

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be less than the limit of quantitation (LOQ) of the analytical method. 

This does not infer that no test material was in solution but that the dissolved concentration (i.e. bioavailable to the test organisms) was below the limit of quantitation which was assessed down to 0.50 mg/L.

The measured test concentration gave an estimate of the dissolved concentration of the test material and hence bioavailable to the test organisms.

During the media preparation trial a test concentration of 1.7 mg/L was attained, however during the definitive test the test material could not be determined in the test media. This was considered to be due to slight differences in stirring speeds and/or slight differences in water quality.

Based on this information the results are based on concentration as % v/v saturated solution. 

Immobilisation data

Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in Table 2.

There was no immobilisation in 20 daphnids exposed to a test concentration of 100 % v/v saturated solution of the test material for a period of 48 hours. Inspection of the immobilisation data gave the following results:

Time (h)	EC50 (% v/v saturated solution)	95% Confidence limits (% v/v saturated solution)
24	>100	-
48	>100	-

The EC50 after 24 and 48 hours exposure was greater than 100 % v/v saturated solution. The No Observed Effect Concentration after 24 and 48 hours exposure was 100 % v/v saturated solution. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

A single daphnid was observed to be immobilised in the control after 48 hours. This was considered to be due to natural causes rather than any toxic effect given that no other immobilisation was observed throughout the test.

Observations on test material solubility

The test media preparations were observed to be clear, colourless solutions throughout the duration of the test.

Physico-chemical measurements

Temperature was maintained at 22 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Temperatures in the control and test vessels were maintained at 22 °C throughout the test which were slightly in excess of the 20 ± 1 °C given in the protocol. This was considered not to affect the results of the test as no significant adverse effects of exposure were observed in the control daphnids throughout the duration of the test and that the temperatures were within the test guideline specification.

                    

Positive Control

Cumulative immobilisation data from the exposure of Daphnia magna to the reference material during the positive control are given in Table 3. The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 1 and 2 (attached).

Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the trimmed Spearman-Karber method (Hamilton et al 1977) at 24 hours and the probit method (Finney 1971) at 48 hours based on the nominal test concentrations gave the following results:

Time (h)	EC50 (mg/L)	95% Confidence limits (mg/L)
3	>3.2*	-
24	0.97	0.85 - 1.1
48	0.70	0.62 – 0.80

*A 3-Hour EC₅₀ value was not calculated as less than 50% immobilisation occurred at this time point.

The No Observed Effect Concentration after 24 and 48 hours were 0.56 and 0.32 mg/L respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

The slope and standard error of the response curve at 48 hours was 11 (SE = 2.3). Due to the unsuitable nature of the data it was not possible to calculate the slope and error of the response curve at 24 hours.

The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC₅₀ value calculated from all positive controls was 0.78 mg/L (sd = 0.22).

Table 1: Cumulative Immobilisation Data in the Range-finding Test

Nominal Concentration (mg/L)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
0.17	0	0
1.7 [1]	0	0

[1] Concentration based on chemical analysis of a saturated solution prepared in a similar manner during the media preparation trial.

Table 2: Cumulative Immobilisation Data in the Definitive Test

Nominal Concentration (% v/v saturated solution)		Cumulative Immobilised Daphnia (Initial Population: 5 Per Replicate)
		24 Hours			48 Hours
		No. Per Replicate	Total	%	No. Per Replicate	Total	%
Control	R1	0	0	0	0	1	1
	R2	0			0
	R3	0			0
	R4	0			1*
100	R1	0	0	0	0	0	0
	R2	0			0
	R3	0			0
	R4	0			0

* Considered to be due to natural causes rather than any toxic effect given that no other immobilisation was observed throughout the test.

R₁– R₄= Replicates 1 to 4

Table 3: Cumulative Immobilisation Data in the Positive Control

Nominal Concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	3 Hours				24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
0.32	0	0	0	0	0	0	0	0	0	0	0	0
0.56	0	0	0	0	0	0	0	0	1	2	3	15
1.0	0	0	0	0	6	5	11	55	9	10	19	95
1.8	0	0	0	0	10	10	20	100	10	10	20	100
3.2	2	1	3	15	10	10	20	100	10	10	20	10

R₁– R₂= Replicates 1 and 2

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, no toxic effects were observed at saturation of the test material. Therefore the 48 hours EC50 was determined to be > 100 % v/v saturated solution and correspondingly the NOEC was 100 % v/v saturated solution.

Executive summary:

The short term toxicity of the test material to Daphnia magna was determined in a GLP study performed according to the standardised guidelines OECD 202 and EU Test Method C2, in an acute immobilisation test.

Pre-study solubility work showed that whilst the test material was readily soluble/dispersable in deionised reverse osmosis water, the test material formed a precipitate.  Following a media preparation trial it was considered appropriate to prepare the test media by the use of a saturated solution using a stirring period of 24 hours followed by filtration (0.2µm, 2 litres discarded to pre-condition the filter) to remove any undissolved test material prior to exposure of the test organisms. Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be less than the limit of quantitation (LOQ) of the analytical method. This does not infer that no test material was in solution but that the dissolved concentration (i.e. bioavailable to the test organisms) was below the limit of quantitation which was assessed down to 0.50 mg/L. The concentration of the test material could not be determined in the test media. Therefore the results are based on concentration as % v/v saturated solution.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a saturated solution of the test material for 48 hours at a temperature of 22 °C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours. A positive control conducted using potassium dichromate, results of which were in the normal range for this reference material.

Under the conditions of the test, no toxic effects were observed at saturation of the test material. Therefore the 48 hours EC50 was determined to be > 100 % v/v saturated solution and correspondingly the NOEC was 100 % v/v saturated solution.

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

July 24 to 26, 2001.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with GLP and agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
100% saturated solution (3.35 mg/l)

- Sampling method:
Not stated.

- Sample storage conditions before analysis:
Not stated.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Stock solution (nominal concentration): 100mg/l, the stock solution was prepared with dilution water.
Dispersion treatment: 24 h magnetic stirrer, filtration with 0.45 µm before use.
Limit concentration: Saturated solution.
DOC: 2.12 mg/l
Calculated concentration: 3.35 mg/l

- Eluate:
Not stated.

- Differential loading:
Not applicable.

- Controls:
test medium control and the reference substance.
Control replicates without test item were tested under the same conditions as the test groups.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
Not applicable.

- Concentration of vehicle in test medium (stock solution and final test solution):
Not applicable.

- Evidence of undissolved material (e.g. precipitate, surface film, etc):
None stated.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name:
Water Flea

- Strain:
STRAUS

- Source:
Institut für Wasser-, Boden- und Luftthygiene des Bundesgesundheitsantes, Corrensplatz, 14195 Berlin, FRG.

- Age at study initiation (mean and range, SD):
2 to 24 hours, obtained by removing the mother animals twice within 22h.

- Method of breeding:
Not stated.

- Feeding during test
Not applcaible.

ACCLIMATION
- Acclimation period:
2 hours.

- Acclimation conditions (same as test or not):
As test.

- Type and amount of food:
Not applicable.

- Feeding frequency:
Not applicable.

- Health during acclimation (any mortality observed):
No mortality observed.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

Theoretical hardnes: CaCO3 = 256mg/l
Equipment; Spectrophotometer, Cadas 100 LPG 158 (Dr. Lange) (water)

Test temperature:

20 ± 1 deg C.
The temperature was recorded throughout the test with a thermohygrograph.
Equipment; Hygro-thermograph (Klimatherm)

pH:

Saturated solution:
At 0 hours: 7.29
At 48 hours: 7.52-7.61 (with all replicates)

Control:
At 0 hours: 7.75
At 48 hours: 7.57-7.62 (with all replicates)
Equipment; pH-Meter inoLab pH Level 1(WTW)

Dissolved oxygen:

Saturated solution:
At 0 hours: 7.81
At 48 hours: 7.93-8.02 (with all replicates)


Control:
At 0 hours: 8.10
At 48 hours: 8.09-8.14 (with all replicates)
Equipment; Oximeter, Oxi 191 (WTW)

Salinity:

Not applicable.

Nominal and measured concentrations:

Nominal concentration: 100% saturated solution
Calculated concentration: 3.35 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable):
closed

- Material, size, headspace, fill volume:
Glass beakers (5cm ID x 8cmH), 50 ml volume will be used.
20 ml volume of the study medium.

- Aeration:
Not stated.

- Type of flow-through (e.g. peristaltic or proportional diluter):
Not applicable.

- Renewal rate of test solution (frequency/flow rate):
Not applicable.

- No. of organisms per vessel:
5-study, 10-reference substance.

- No. of vessels per concentration (replicates):
4

- No. of vessels per control (replicates):
2-reference substance.
4-test medium control

- No. of vessels per vehicle control (replicates):
Not applicable.

- Biomass loading rate:
Not stated.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
As stated in EEC 92/69 L383A C.2 (Annex)

- Total organic carbon:
Not stated.

- Particulate matter:
Not stated.

- Metals:
Not stated.

- Pesticides:
Not stated.

- Chlorine:
Not stated.

- Alkalinity:
Not stated.

- Ca/mg ratio:
Not stated.

- Conductivity:
Not stated.

- Culture medium different from test medium:
Not stated.

- Intervals of water quality measurement:
48 hours.


OTHER TEST CONDITIONS
- Adjustment of pH:
Not stated.

- Photoperiod:
16 hours light/8 hours dark

- Light intensity:
20 µmol.m-2.S-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The percentage immobility was determined in all test and control group after 24 h and 48 h.


TEST CONCENTRATIONS
- Spacing factor for test concentrations:
Not applcable.

- Justification for using less concentrations than requested by guideline:
Not applcable.

- Range finding study
Not applcable.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate p.a.

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 3.35 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Prepared as a 100 % saturated solution

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 100 % saturated solution

Details on results:

- Behavioural abnormalities:
Not stated.

- Observations on body length and weight:
Not stated.

- Other biological observations:
The percentage immobility was determined in the tested limit concentration and the control after 24 h 48 h. The immobilisation rates in % after 24 h and 48 h of exposure are given in Table 2. There is no biologically significant effect neither in the tested limit concentration nor in control group.

- Mortality of control:
None.

- Other adverse effects control:
Not stated.

- Abnormal responses:
Not stated.

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
Not stated.

- Effect concentrations exceeding solubility of substance in test medium:
The test item was clearly dissolved in the saturated solution after filtration with 0.45 µm throughout exposure.

Results with reference substance (positive control):

- Results with reference substance valid?
The percentage immobility for the reference item was determined after 24 h. The EC100 value was determined from the test results. The EC10 and EC50 with 95% confidence interval (CI) were determined by probit analysis according to standard procedures.

Ec- values after 24 h of the reference item in mg/l were as follows:
EC10: 1.3
EC50: 2.0 (CI 1.8 -2.2)
EC100: 5.8


- Mortality:
Not stated.

- EC50/LC50:
EC50: 2.0 (CL 1.8-2.2)

- Other:
The EC50 - value of reference item potassium dichromate after 24 h is within the prescribed concentration range of 1.0-2.5 mg/l of quality criteria according to AQS: DIN Guideline 38412 L 30.

Table 2: Percentage number of daphnids incapable of swimming after 24 and 48 h of exposure (n=20)

Immobilisation [%]
Limit Conc.	24 h					48 h
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
Saturated solution	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

MV=Mean value

 

Water parameters.

The water parameters (dissolved oxygen concentrtion, pH-value) of test and control groups at the beginning of the test in one replicate per concentration and control and in all replicates after 48 h are listed in Tables 3-4 attached.

pH-value, oxygen concentration, temperature, conductivity and total hardness of the dilution water were measured prior to test start. The results are listed in Table 5 attached.

 

Validity criteria fulfilled:

yes

Remarks:

All quality criteria as listed above were fulfilled.

Conclusions:

In the tested saturated solution (DOC = 2.12 mg/l) no biologically significant effect was determined. The concentration of the tested limit concentration was calculated by DOC analysis to be 3.35 mg/l, therefore 48h EC50>3.35mg/l.

Executive summary:

The toxicity of the test material was determined in an acute immobilisation test with Daphnia magna (STRAUS). The effect of a saturated solution of the test material was determined according to OECD Guideline 202 and EU Method C.2.

The test solutions were prepared using a loading rate of 100 mg/L. Any undissolved material was removed by filtration prior to use. The study was performed as a limit test.

The study was conducted under static conditions over a duration of 48 hours. 20 test organisms were exposed to the test material and control.

The test material was analysed in solution by DOC analysis. In the tested saturated solution (DOC = 2.12 mg/L arithmetic mean measured concentration) no biologically significant effect was determined. The calculated concentration of the test material in solution was 3.35 mg/L.

No immobilisation of the Daphnia was observed during the test; therefore the EC50 is greater 3.35 mg/L, than the limit of solubility of the test material.

Reference 4

Endpoint:

short-term toxicity to aquatic invertebrates

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

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 39.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

26.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: (95 % CI: 22.41 to 31.57 mg/L)

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 39.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

22.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

39.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Reference 5

Endpoint:

short-term toxicity to aquatic invertebrates

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

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 other: % v/v

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Description of key information

Supporting Study on Read Across Substance GS-11P-T: Sipos (2017)
Under the conditions of the study the 48-h EC50 was 26.60 mg/L (measured) with 95 % CI of 22.41 to 31.57 mg/L. The 48-h EC100 was > 39.60 mg/L, the 48-h NOEC was 22.64 mg/L and the 48-h LOEC was 39.60 mg/L.

Supporting Study on the Target Substance: Noack (2001)
48 h EC50 >3.35 mg/L (arithmetic mean measured concentration), 48 h EL50 > 100 mg/L (nominal) loading rate (100 % (v/v) saturated solution), OECD 202, EU Method C.2.

Key Study on Read Across Substance HS-21P-T: Goodband & Mullee (2009)
Under the conditions of the test, no toxic effects were observed at saturation of the test material. Therefore the 48 hours EC50 was determined to be > 100 % v/v saturated solution and correspondingly the NOEC was 100 % v/v saturated solution.

Additional information

Supporting Study on Read Across Substance GS-11P-T: Sipos (2017)

In the supporting study on read across substance GS-11P-T Sipos (2017). the acute toxicity of the test material was investigated in accordance with the standardised guidelines OECD 202, EU Method C.2, EPA OCSPP 850.1010 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).

Testing was performed on Daphnia magna in an acute immobilisation test, over an exposure period of 48 hours in a semi-static test system.

Even though the test material seems to be soluble in the test media, strong precipitation was observed within minutes. For this reason the experiment was carried out in a different media. A 1:1 v/v solution of ISO medium and deionised water was used as a test medium instead of ISO medium.

Because significant immobility 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 test under semi-static conditions.

The nominal concentrations of test material used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solution.

The test concentrations were analytically determined at the start and at the end of the renewal periods. The corresponding measured geometric mean test material concentrations were: 3.56; 5.48; 11.19; 22.64 and 39.60 mg/L. Biological results are based on the measured geometric mean test material concentrations.

Twenty animals divided into four groups of five animals each were used at the test concentrations and for the control as well.

The 24 hours EC50 values of the test material could not be calculated due to the slight effects observed. The 48 hours EC50 values were calculated using Probit analysis by TOXSTAT software. The 24 h EC50 and 48 h EC100 values of the test material were determined directly from the raw data. For the determination of the LOEC and NOEC, the immobilisation at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software.

All validity criteria were met during this study.

Under the conditions of the study the 48-h EC50 was 26.60 mg/L with 95 % CI of 22.41 to 31.57 mg/L. The 48-h EC100 was > 39.60 mg/L, the 48-h NOEC was 22.64 mg/L and the 48-h LOEC was 39.60 mg/L.

 

Supporting Study on the Target Substance: Noack (2001)

In the key study (Noack 2001), the toxicity of the test material was determined in an acute immobilisation test with Daphnia magna (STRAUS). The effect of a saturated solution of the test material was determined according to OECD Guideline 202 and EU Method C.2. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality as defined in Klimisch et al. (1997).

The test solutions were prepared using a loading rate of 100 mg/L. Any undissolved material was removed by filtration prior to use. The study was performed as a limit test.

The study was conducted under static conditions for a duration of 48 hours. 20 test organisms were exposed to the test material and control.

The test material was analysed in solution by DOC analysis. In the tested saturated solution (DOC = 2.12 mg/L arithmetic mean measured concentration) no biologically significant effect was determined. The calculated concentration of the test material in solution was 3.35 mg/L.

No immobilisation of the Daphnia was observed during the test; therefore the EC50 is greater 3.35 mg/L, the limit of solubility of the test material.

 

Key Study on Read Across Substance HS-21P-T: Goodband & Mullee (2009)

In the supporting study on target substance HS-21P-T Goodband & Mullee (2009), the short-term toxicity of the test material to Daphnia magna was determined in a GLP study performed according to the standardised guidelines OECD 202 and EU Test Method C2, in an acute immobilisation test. The study was conducted in compliance with GLP and agreed protocols and was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Pre-study solubility work showed that whilst the test material was readily soluble/dispersable in deionised reverse osmosis water, the test material formed a precipitate.  Following a media preparation trial it was considered appropriate to prepare the test media by the use of a saturated solution using a stirring period of 24 hours followed by filtration (0.2µm, 2 litres discarded to pre-condition the filter) to remove any undissolved test material prior to exposure of the test organisms. Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be less than the limit of quantitation (LOQ) of the analytical method. This does not infer that no test material was in solution but that the dissolved concentration (i.e. bioavailable to the test organisms) was below the limit of quantitation which was assessed down to 0.50 mg/L. The concentration of the test material could not be determined in the test media. Therefore the results are based on concentration as % v/v saturated solution.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a saturated solution of the test material for 48 hours at a temperature of 22 °C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours. A positive control conducted using potassium dichromate, results of which were in the normal range for this reference material.

Under the conditions of the test, no toxic effects were observed at saturation of the test material. Therefore the 48 hours EC50 was determined to be > 100 % v/v saturated solution and correspondingly the NOEC was 100 % v/v saturated solution.