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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to protocol.
Justification for type of information:
The data is read across from a similar substance in a category approach.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
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 6 occasions. All samples were diluted with leaching solution.

At least 10 mL was sampled in each case this was diluted 1:1 with leaching solution (see for details Appendix 4). 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 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 at room temperature in the dark until analysis, or analyzed immediately depending upon available storage space and logistical possibilities during the study.
Vehicle:
no
Details on test solutions:
PREPARATION OF STOCK SOLUTIONS
The test substance is readily soluble/dispersible in water. To prepare the stock solutions for every water renewal, on average 0.2048 g (standard deviation: 0.0017 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 in an attempt to completely dissolve the test substance. The pH of each stock solution was checked and found to be between 7.9 and 8.1 and was therefore not adjusted. After this the stock solution was filled up to 100 mL with test medium.
A fresh stock solution was prepared for each solution change.

PREPARATION OF TEST SOLUTIONS
Test solutions were prepared by further dilution of the stock solution with M4.
A geometric series of concentrations was used. The ratio between two consecutive concentrations was 2.
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 fresh M4 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). The culture is checked yearly for sensitivity by a reference test with potassium dichromate.

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:
Not measured
Test temperature:
19.8 to 21.0 °C.
pH:
7.5 to 8.8
Dissolved oxygen:
8.3 to 10.5 mg O2/L.
Nominal and measured concentrations:
Nominal and measured concentrations: 0, 3.25, 7.5, 15, 30 and 60 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL (nominal) glass beakers were used, containing approximately 50 mL of test solution and covered by glass plates during the test.
- Type (delete if not applicable): open
- Aeration: no
- Renewal rate of test solution: 3 times per 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
- Source/preparation of dilution water: Elendt M4 medium
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16h light
- Light intensity: 18.3 µmol/s/m2

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Animals were checked for immobilisation of parent daphnids on at least 6 days per week of the test, by gently shaking the test vessel. From the day of the first brood, observations of broods (aborted, living and dead progeny) were also made in each container at each concentration. The day of brood release and the number of living and dead neonates per brood or abortions were noted. Any other observations of abnormalities were also recorded.
At the end of the test, the length of all surviving parent animals was measured to the nearest 0.1 unit using a binocular microscope (One unit equals 1.5 mm). Following measurement of body length, parent animals were placed in an oven at 105 °C overnight in groups and weighed (to constant dry weight) to calculate the mean individual weight per surviving parent animals per concentration.

RANGE-FINDING STUDY
Based on an extended second brood range finding test in unfiltered natural river water and an acute range finding test in synthetic medium a 9d-NOEC for reproduction of 24 mg/L was calculated. The EC50 in the acute test was not reached and was therefore greater than 100 mg/L, the NOEC for immobility was 10 mg/L.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
13.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: weight
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
6.9 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: weight
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
28.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
32.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: length
Remarks on result:
other: 95% C.I. 3.8 -44.9 mg/L
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
15 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: reproduction and length
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
30 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: reproduction and length
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 60 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
parent animals
Details on results:
CHEMICAL ANALYSIS
The results of measured concentrations in the test series were all above 80% of the nominal concentrations.
Also the stock solutions used to make the test solutions showed a good recovery. Because the measured concentrations were all close to nominal, nominal test substance concentrations are used for endpoint calculations.

OTHER BIOLOGICAL EFFECTS
The first brood was in most cases released on day 7 to 8. In the highest test concentration of 60 mg/L first brood of the parents still alive was produced on day 10.
Dead neonates were produced in 30 and 60 mg/L. In 30 mg/L 9 out of 10 parent daphnids produced dead offspring in several occasions. Two animals had some aborted eggs in one occasion. In 60 mg/L one animal produced one dead young and two animals had some aborted eggs in one occasion.

Results with reference substance (positive control):
The results of this test were within the range given in the relevant guideline.
Reported statistics and error estimates:
The data on reproduction and length was 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 using Dunnett’s test.
An EC10and EC50 was determined applying probit analysis using Toxcalc.
An EC50 based on survival of parent animals at the end of the test was determined applying probit analysis Toxcalc.
Validity criteria fulfilled:
yes
Conclusions:
The test is reliable because it is performed according to standard OECD guideline 211 under GLP condition and all validity criteria were fulfilled. The test substance was stable during the test and >80% of the nominal test substance concentration was measured. Therefore nominal test concentrations were used for the dose-response curve. The calculated endpoints are therefore adequate for C&L and risk assessment purposes.
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, 3.25, 7.5, 15, 30 and 60mg/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 206after 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.

 

Parental weightwas found to be the most sensitive endpoint based on the EC10-values.

An EC10for parental weight was determined as 13.1 mg/L. The EC10for reproduction was 28.2 mg/L. Parental length gave an EC10of 32.8 mg/L with 95% confidence limits of 3.8 to 44.9 mg/L.

Using Dunnett’s test, the Lowest Observed Effect Concentration (LOEC) was found to be 30 mg/L for reproduction and parental length. Based on these statistical results the NOEC is 15 mg/L.

The EC50for parent survival was greater than 60 mg/L.

Description of key information

The long term toxicity of Naphthalenesulfonic acid, methyl, butyl, sodium salt to daphnia is read across from the structurally related substance Aromatic Hydrocarbons, C10-13, Reaction Products With Branched Nonene Sulphonated.

An acute daphnia rangefinding test has been performed to evaluate which of two very similar alkylnaphthalene sulphonates is the more toxic substance. The results obtained indicate that there is a slightly higher toxicity observed for batch with a higher content of longer alkyl chain derivative when compared to the one with a higher content of shorter alkyl chains.

This observation supports the read across of the acute fish and long term daphnia test from the source chemical Aromatic Hydrocarbons, C10-13, Reaction Products With Branched Nonene Sulphonated, sodium salt to the structurally strongly related target chemical Naphthalenesulfonic acid, methyl, butyl, sodium salt.

The 21d-EC10 Daphnia magna performed with the batch with a higher content of longer alkyl chains was 6.9 mg/L in a OECD TG 211 Daphnia magna reproduction test under GLP (Reliability 1) based on active ingredient concentration.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
6.9 mg/L

Additional information

The long term toxicity of Naphthalenesulfonic acid, methyl, butyl, sodium salt to daphnia is read across from the structurally related substance Aromatic Hydrocarbons, C10-13, Reaction Products With Branched Nonene Sulphonated.

The toxicity of Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts to Daphnia magna was tested according to OECD TG 211 Daphnia magna reproduction test. Parental weight was the most sensitive parameter. EC10 for parental weight was reported to be 13.1 mg/L in the 21 day test under GLP conditions. This value equals to 6.9 mg/l based on the active ingredient concentration. The definitive daphnia test has been performed with the high nonene naphthalene sulphonate because algae and daphnia range finding tests with both the low nonene and high nonene substance indicated a slightly higher toxicity for the high nonene naphthalene sulphonate.

As the results of both range finding tests are considered to be valid and because both test substances are very similar in their fate during these tests, the conclusion that the high nonene derivative is slightly more toxic than the low nonene derivative is considered sufficient to select the more toxic derivative as the worst-case representative in the hazard assessment of both substances. For this reason read-across of ecotoxicity results of the source chemical (the high nonene derivative) to the target chemical (the low nonene derivative) is considered to be justified without the introduction of an additional safety factor.

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