L-Glutamic acid, N-coco acyl derivs., compds. with triethanolamine (1:1)

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 August 2017 to 11 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

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: JMAFF 2-7-2-1

Version / remarks:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

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

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 levels at the beginning and at the end of the renewal periods.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- A stock solution with a concentration of 100.0 mg/L (nominal) was prepared with direct addition of the test material, mixed into the test medium (ISO Medium) using ultrasonic bath (approximately 10 minutes). The test solutions were prepared by appropriate dilution of this stock solution just before introduction of the Daphnia (start of the treatments).
- Because the test material is not stable over the 48h 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 the treatment, at each of the renewal periods test solution was prepared by the method described above.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Breeding: The Daphnia are bred in Ecotoxicological Laboratory of CiToxLAB Hungary Ltd. The health of the stock animals is continuously monitored by visual daily checking. Abnormal behaviour or significant decrease of population is recorded.
- Age: They were 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:

249 mg/L (as CaCO3)

Test temperature:

20.5– 20.8 °C

pH:

7.26 – 7.66

Dissolved oxygen:

7.7 – 8.6 mg/L

Nominal and measured concentrations:

- Nominal concentrations: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L.
- Measured geometric mean concentrations: 7.11, 14.63, 29.35, 58.19 and 114.61 mg/L.
- As the measured concentrations deviated more than 20 per cent from the nominal in some cases, the biological results are based on the measured geometric mean test material concentrations

Details on test conditions:

TEST SYSTEM
- Test vessels: Glass beakers
- Material, size, headspace, fill volume: At least 5 mL test solution/animal.
- Renewal rate of test solution: The frequency of the water renewal periods was every 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4

TEST MEDIUM / WATER PARAMETERS
- Reconstituted water (ISO medium, according to OECD 202) was used as dilution water for both the range finding and definitive tests. The same composition of reconstituted water was used for the tests and for breeding the test animals.
- Source/preparation of dilution water: Separate stock solutions of individual trace elements were first prepared in deionised water. The medium was prepared of these different stock solutions, so it contained all trace elements (combined solution). Components of medium: CaCl2.2H2O 11.76 g/L 25 mL/L, MgSO4.7H2O 4.93 g/L 25 mL/L, KCl 0.23 g/L 25 mL/L and NaHCO3 2.59 g/L 25 mL/L.
- Culture medium different from test medium: No
- Intervals of water quality measurement: The water temperature, the oxygen concentrations and pH of the controls and the test solution were measured at the beginning and at the end of the renewal periods.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16-hour light and 8-hour dark cycle

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.

RANGE-FINDING STUDY
- Test concentrations (nominal): 0.1, 1, 10, and 100 mg/L
A concentration range-finding test was conducted to determine the approximate toxicity of the test item 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.
The test solutions were prepared by appropriate dilution of the stock solution.
- Results used to determine the conditions for the definitive study: Yes, the choice of the test concentration was made on the basis of the results of the preliminary range-finding tests. At 100 mg/L 8 out of 10 daphnids treated were immobilised. Because significant immobility was observed at the examined concentration levels during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.0) and one control were used in the main experiment 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 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

104.69 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: (95 % conf. limits: 96.73 – 113.31 mg/L)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

67.21 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: (95 % conf. limits: 62.40 – 72.40 mg/L)

Key result

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 114.61 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.:

29.35 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.:

58.19 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 mg/L. The corresponding measured geometric mean test material concentrations were: 7.11, 14.63, 29.35, 58.19 and 114.61 mg/L.
- As the measured concentrations deviated more than 20 per cent from the nominal in some cases, the biological results are related to the geometric mean of the measured test material concentrations.

IMMOBILISATION
- The number of immobilised animals and the percentage of immobility were determined at the 24th and 48th hour (Table 4).
- In addition to immobility, no abnormal behaviour or appearance of test animals was detected.

Results with reference substance (positive control):

- The 24h EC50: 0.64 mg/L, (95 % confidence limits: 0.59 – 0.68 mg/L)

Reported statistics and error estimates:

- The 24 and 48 hours EC50 values of the test material were calculated using Probit analysis by TOXSTAT software. The 48 hours 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.

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.

Table 1: Measured concentrations of the first renewal period with the 95 % confidence intervals

Sample concentration (mg/L)	Measured concentration at the start (mg/L)	Measured concentration at the end (mg/L)
Control	n.d.	n.d.
6.25	6.97 ± 0.115	7.29 ± 0.160
12.5	14.2 ± 0.13	15.3 ± 0.19
25	28.6 ± 0.10	30.9 ± 0.47
50	56.7 ± 0.51	61.1 ± 1.41
100	112 ± 2.2	120 ± 2.3

 

Table 2: Measured concentrations of the second renewal period with the 95 % confidence intervals

Sample concentration (mg/L)	Measured concentration at the start (mg/L)	Measured concentration at the end (mg/L)
Control	n.d.	n.d.
6.25	6.66 ± 0.108	7.57 ± 0.212
12.5	13.5 ± 0.40	15.6 ± 0.20
25	27.0 ± 0.23	31.1 ± 0.26
50	53.8 ± 0.12	61.5 ± 1.40
100	107 ± 1.8	120 ± 2.2

 

Table 3: Calculation of exposure concentrations

Nominal concentration (mg/L)	Measured concentration 1^strenewal period (mg/L)		Measured concentration 2^ndrenewal period (mg/L)		Geometric mean (mg/L)
Control	n.d.	n.d.	n.d.	n.d.	-
6.25	6.97	7.29	6.66	7.57	7.11
12.5	14.2	15.3	13.5	15.6	14.63
25	28.6	30.9	27.0	31.1	29.35
50	56.7	61.1	53.8	61.5	58.19
100	112	120	107	120	114.61

   

Table 4: Number and percentage of immobilised animals

Concentration (mg/L)		Number of treated animals	Immobilised animals
Nominal	Measured		24 hours		48 hours
			number	percent	number	percent
Control	0.00	20	0	0	0	0
6.25	7.11	20	0	0	0	0
12.5	14.63	20	0	0	0	0
25	29.35	20	0	0	0	0
50	58.19	20	1	5	8	40*
100	114.61	20	12	60*	18	90*

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

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study the 48-h EC50 was 67.21 mg/L (95 % confidence limits: 62.40 – 72.40 mg/L).

Executive summary:

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

Because significant immobility was observed at the examined concentration levels during the preliminary range-finding tests, 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 test concentrations were analytically determined at the start and at the end of the renewal periods.

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. The corresponding measured geometric mean test item concentrations were: 7.11, 14.63, 29.35, 58.19 and 114.61 mg/L.

As the measured concentrations deviated more than 20 per cent from the nominal in some cases, biological results are based on the measured geometric mean test item concentrations.

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

The 24 and 48 hours EC50 values of the test item were calculated using Probit analysis by TOXSTAT software. The 48h EC100 values of the test item 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 this study the 48-h EC50 was 67.21 mg/L (95 % confidence limits: 62.40 – 72.40 mg/L). The 24-h EC50 value was 104.69 mg/L (95 % conf. limits: 96.73 – 113.31 mg/L) and the 48-h EC100 value was > 114.61 mg/L. The 48-h No-Observed Effect Concentration (NOEC) was 29.35 mg/L and the 48-h Lowest Observed Effect Concentration (LOEC) was 58.19 mg/L.

Description of key information

Under the conditions of this study the 48-h EC50 was 67.21 mg/L (95 % confidence limits: 62.40 – 72.40 mg/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

67.21 mg/L

Additional information

The acute toxicity of the test material on Daphnia magna was assessed in an acute immobilisation test, over an exposure period of 48 hours in a semi-static test system. The test was performed in accordance with the standardised guidelines OECD 202, EPA OCSPP 850.1010 and JMAFF 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).

Because significant immobility was observed at the examined concentration levels during the preliminary range-finding tests, 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 test concentrations were analytically determined at the start and at the end of the renewal periods.

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. The corresponding measured geometric mean test item concentrations were: 7.11, 14.63, 29.35, 58.19 and 114.61 mg/L.

As the measured concentrations deviated more than 20 per cent from the nominal in some cases, biological results are based on the measured geometric mean test item concentrations.

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

The 24 and 48 hours EC50 values of the test item were calculated using Probit analysis by TOXSTAT software. The 48h EC100 values of the test item 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 this study the 48-h EC50 was 67.21 mg/L (95 % confidence limits: 62.40 – 72.40 mg/L). The 24-h EC50 value was 104.69 mg/L (95 % conf. limits: 96.73 – 113.31 mg/L) and the 48-h EC100 value was > 114.61 mg/L. The 48-h No-Observed Effect Concentration (NOEC) was 29.35 mg/L and the 48-h Lowest Observed Effect Concentration (LOEC) was 58.19 mg/L.