N-(2-carboxyethyl)-N-octyl-β-alanine

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to protocol.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

Water hardness was measured only once at the start of the test in the dilution medium as only one batch was used for the test.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples of all the test concentrations were taken on preparation, just before and after changing of the test solution.

At least 10 mL was sampled in each case this was diluted 1:1 with leaching solution. The samples were then gently shaken and a 1 mL sample was then transferred into the analytical vials for analysis. With each transfer step the pipettes used were rinsed with the solution to be analysed and the solution discarded before sampling for the actual analysis. This helps to minimise loss to pipette surfaces etc. Samples from the actual test replicates were filtered using a 0.45 µm GHP Acrodisc filter to remove algae. Filters were primed with the relevant solution before use to minimise the loss through sorption.
The samples were stored in leaching solution in the refrigerator in the dark until analysis, or analyzed immediately depending upon available storage space and logistical planning during the study.
Stability samples were stored during the first definitive study. After 2 and 7 days of storage these samples were reanalysed and compared to freshly prepared samples.

Vehicle:

no

Details on test solutions:

Preparation of the stock solutions:
The test substance is readily soluble in water. To prepare the stock solutions for every water renewal, on average 0.1232 g (standard deviation: 0.0024 g) of test substance was weighed on an analytical balance then dissolved directly in approx. 80 mL test medium. The stock solutions were agitated mechanically for approximately 30 minutes to completely dissolve the test substance. The pH of each stock solution was checked and found to be between 9.0 and 9.1 and was therefore adjusted with 1 M HCl to 7.9 – 8.1. After this the stock solution was filled up to 100 mL with test medium.
Once a stock solution of 1.240 g in 1000 mL was prepared. On one occasion the stock solution had a pH of 9.1 and was erroneously not adjusted. As all pH levels of the test concentrations were 8.0 - 8.2, this did not have an impact on the outcome of the test.
A fresh stock solution was prepared for each solution change.

Preparation of the test solutions:
Test solutions were prepared by further dilution of the stock solution with HD water.
A geometric series of concentrations was used. The ratio between two consecutive concentrations was 2.5.
Test vessels were filled directly from the test solution containers immediately after preparation. The solutions were renewed three times a week during the test.

The pH of the test solutions were all between 6 and 9 at refreshment and close to the value of the test medium.
One control containing only test medium was included in the test.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test animals were taken from a Daphnia magna clone 5 stock, (Origin: NOTOX B.V The Netherlands) cultured in conformity with the relevant SOP. The animals used in the test were less than 24 hours old and were obtained from parent animals reproducing parthenogenically and having an age of 2-4 weeks (having previously produced at least one brood before use).

Culture animals are fed a diet of 0.1 to 0.2 mg of carbon per daphnid per day, in the form of the algal strain Chlorella vulgaris. The strain is cultured in the Environmental Chemistry laboratory and total organic carbon content has previously been measured.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

5.73 °dH
Ca 37.01 mg/L

Test temperature:

19.7 - 21.2 °C

pH:

7.6 - 9.3
The higher values were only seen in the first days of the test as parent daphnids are still small and do not eat all algae present. Due to algae growth pH levels can increase. As no animals died and performed well this is not considered to have an impact on the outcome of the test.

Dissolved oxygen:

7.5 - 11.3 mg O2/L
The high oxygen concentrations were found in the first days of the test and were again due to algae growth.

Nominal and measured concentrations:

Nominal: 1.6, 4, 10, 25 and 62.5 mg/L
TWA: 1.6, 3.7, 9.0, 22.0, 54.7 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL glass beakers
- Aeration: no
- Renewal rate of test solution: 3 times a week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
Natural surface water was used as test medium.
Natural surface water, called Heveadorp water, was sampled in a small brook at the Fonteinallee in Doorwerth. This sample point has a historically low DOC and TSS concentration.
Natural surface water was filtered through a Whatman 589/2 filter in order to eliminate the suspended solids and was frozen afterward until use. The composition of the chosen surface water is detailed in the table below.

Characteristics of Heveadorp water (HD water):
- Sampling Date: June 20, 2012
- Weather: Moderate overcast
- Appearance: Clear yellowish
- pH: 8.1
- Conductivity (µS/cm): 317
- DOC (mg/L): 2 (not measured under GLP)
- Ammonium- N (mg/L): 0.018
- Total Phosphate (mg/L): 0.011
- Total hardness (ºdH) 5.73
(mg/L) Ca 37.01
1 Below the recommended minimum value of 140 mg/L

Water hardness was lower than recommended in the guideline. But because the daphnids showed excellent performance in this test medium and from literature it is also known that D. magna is tolerant to low levels of water hardness. Therefore, it is not considered to have an impact on the outcome of the test.

OECD recommended M4 media vitamins were added to HD water to create the test medium. This was done in the normal manner as described for Elendt M4 medium. Test media was aerated prior to use with filtered air.

- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: yes, of the stock solutions
- Photoperiod: 16 hours light
- Light intensity: 17.4 µmol/s/m2

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : reproduction, parental length and parental weight.

RANGE-FINDING STUDY
- Results used to determine the conditions for the definitive study: Extended first brood range finding test in synthetic medium where no negative effects on reproduction were found up to 5 mg/L and significant effects on reproduction at 10 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 62.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: equivalent to > 24.06 mg a.i./L

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 62.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: parental length

Remarks on result:

other: equivalent to > 24.06 mg a.i./L

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 62.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: parental weight

Remarks on result:

other: equivalent to > 24.06 mg a.i./L

Details on results:

Analytical results:
See attached illustration for measured concentrations.

Influence of filtration of the test samples was checked in the first definitive study which had to be terminated before day 21. As the measured concentration of the filtered sample was close to the one of the sample which was not filtered, it can be considered that there is no loss due to filtration and no corrections need to be made for filtration.

Storage stability was checked at the beginning of the test. Samples taken at the start and at day 2 of the study were analyzed immediately after sampling, then stored in the refrigerator for 2 and 7 days, respectively and analyzed again. All measured concentrations stayed within 80 to 120% of the nominal values and storage stability was found satisfactory and no further stability controls were included in the study.

The so called Qualifier and Quantifier were analyzed. The Quantifier is used for quantification of the test substance, the Qualifier is used for confirmation of the identity of the test substance. All measured concentrations given in the report are therefore based on the Quantifier data.
Stability samples were analyzed after 2 and 7 days of storage and were found within the limits set. Stability was proven this way and no more stability samples were included when samples were stored before analysis.

Biological results:
Parent animal mortality
No parental mortality was found in any of the concentrations tested or the control.

Coefficient of variation of control fecundity
The validity criterion for the coefficient of variation (less than 25% in the control based on the number of living neonates for each parent animal alive at the end of the test) was achieved.

The data on reproduction was tested for normality using Kolmogorov D test and found to be normally distributed. The data passed Bartlett’s tests for homogeneity of variance. Analysis of variance was performed on the number of living neonates per parent alive using Dunnett’s test.

No significant effect on reproduction compared to the control were observed in any of the test concentrations, therefore the Lowest Observed Effect Concentration (LOEC) is greater than 62.5 mg/L and No Observed Effect Concentration (NOEC) is greater than or equal to 62.5 mg/L.

Further statistical analyses were performed using length and weight data.
Length data were not found to be normally distributed, but displayed homogeneity of variance. For weight data however normality and homogeneity of variance could not be confirmed because animals were pooled in only two groups per test concentration to be able to weigh accurately. Analysis of variance was performed on parental length and weight using Dunnett’s test. For both endpoints no significant differences were found compared to the control in any of the test concentrations.
The LOEC and NOEC after 21 days based on parental length and weight are respectively > 62.5 and ≥ 62.5 mg/L.

EC50 for parent animals
An EC50 based on survival of parent animals at the end of the test could not be determined because no mortality occurred in any of the test concentrations.

Any other biological effects observed
The first brood was in almost all cases released on day 7 for all test concentrations and the control.
A few dead neonates were produced; 1 in the control and in 1.6 mg/L, 2 in 10 and 62.5 mg/L and 3 in 4 and 25 mg/L.

Results with reference substance (positive control):

The culture is checked yearly for sensitivity by a reference test with Potassium dichromate. The results of this test were within the range given in the relevant guideline.

Reported statistics and error estimates:

The data on reproduction, length and weight were tested for normality using Kolmogorov D test. Bartlett’s tests was used for homogeneity of variance. Analysis of variance was performed on the number of living neonates per parent alive, lenght and weight of parents using Dunnett’s test.
Toxcalc was used for all computations. 

Validity criteria fulfilled:

yes

Conclusions:

The study was performed according to protocol under GLP and is therefore valid without restrictions (reliability 1). The results can be used for classification and labelling and risk assessment.

Executive summary:

The purpose of this study was to assess the toxicity of the test substance dissolved in fresh water, on the reproductive efficacy of Daphnia magna STRAUS - clone 5, in a semi-static test complying with the OECD Guideline No. 211

 

The test criterion of toxicity used was reproductive capacity expressed as the number of neonates per daphnid per day.

 

The nominal concentrations used in the study were as follows: 0,1.6, 4, 10, 25 and 62.5 mg/L

All concentrations given refer to the technical product as supplied by the sponsor.

 

Analytical determinations of the test solutions were made on 6 occasions during the test.

The results of the chemical analyses show that the concentrations in the test solutions fresh and used are close to the nominal concentration. All measured concentrations stayed within 80 to 120% of the nominal. Therefore, nominal test substance concentrations are used to calculate the effects.

 

The validity criteria were respected:

No mortality occurred in the control group over the test period.

The average number of juveniles per parent in the control was 155 after 21 days.

 

The No Observed Effect Concentration (NOEC) is determined as the concentration used in the study that is immediately below the Lowest Observed Effect Concentration (LOEC), the latter derived statistically from the data using the appropriate statistical test.

EC₁₀-and EC₅₀-values were determined where possible, using statistical analysis.

 

No significant effects were found up to the highest test concentration on reproduction, parental length or weight using Dunnett’s test.

Therefore, the LOEC was found to be greater than 62.5 mg/L and the NOEC is greater than or equal to 62.5 mg/L.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Justification for type of information:

See read-across justification attached in Section 13.

Reason / purpose for cross-reference:

read-across source

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 62.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: equivalent to > 24.06 mg a.i./L

Description of key information

The NOEC (21 d) reported for an analogue test item was > 24.06 mg a.i./L based on reproduction of Daphnia magna (OECD 211).

Additional information

The purpose of this study was to assess the toxicity of an analogue test substance dissolved in fresh water, on the reproductive efficacy of Daphnia magna STRAUS - clone 5, in a semi-static test complying with the OECD Guideline No. 211

 

The test criterion of toxicity used was reproductive capacity expressed as the number of neonates per daphnid per day.

 

The nominal concentrations used in the study were as follows: 0,1.6, 4, 10, 25 and 62.5 mg/L. All concentrations given refer to the technical product as supplied by the sponsor.

 

Analytical determinations of the test solutions were made on 6 occasions during the test. The results of the chemical analyses show that the concentrations in the test solutions fresh and used are close to the nominal concentration. All measured concentrations stayed within 80 to 120% of the nominal. Therefore, nominal test substance concentrations are used to calculate the effects.

The validity criteria were respected: No mortality occurred in the control group over the test period. The average number of juveniles per parent in the control was 155 after 21 days.

 

The No Observed Effect Concentration (NOEC) is determined as the concentration used in the study that is immediately below the Lowest Observed Effect Concentration (LOEC), the latter derived statistically from the data using the appropriate statistical test. EC10-and EC50-values were determined where possible, using statistical analysis.

 

No significant effects were found up to the highest test concentration on reproduction, parental length or weight using Dunnett’s test.

Therefore, the LOEC was found to be > 62.5 mg/L and the NOEC is ≥ 62.5 mg/L (equivalent to 24.06 mg a.i./L).