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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental starting date: 13-5-2014 Experimental completion date: 15-5-2014
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)
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
The test substance, project sample code T 14009 was supplied by the sponsor. Data on handling, stability, composition, purity or other characteristics of the test substance supplied by the sponsor have been used without further verification.
All concentrations given in this report refer to the technical product as supplied by the sponsor.
Stability: Hydrolytically stable (AN 2014)
Water solubility: 815 mg/L
Storage: -25°C
Batch/Lot: 1205442102
Analytical monitoring:
yes
Remarks:
High Performance Liquid Chromatography (HPLC)
Details on sampling:
Test concentrations
4.0, 8.8, 19.4, 42.5, and 93.7 mg/L
Vehicle:
no
Details on test solutions:
Test solutions:
The test substance is sufficiently soluble in water. To prepare the stock solution 0.0564 g of test substance was weighed on an analytical balance and then dissolved directly in approximately 450 mL of test medium. The stock solution was agitated mechanically for approximately 45 minutes to completely dissolve the test substance. The pH of the stock solution was checked and found to be 8.0 and was therefore not adjusted. After this the stock solution was filled up to 500 mL with test medium.
A clear stock solution with a nominal concentration of 113 mg/L was obtained.

Test concentrations:
Further dilution of the stock solution in test medium was carried out to achieve the following test concentrations:
4.0, 8.8, 19.4, 42.5, and 93.7 mg/L
Test organisms (species):
Daphnia magna
Details on test organisms:
The test animals were taken from a Daphnia magna stock, cultured in conformity with Standard Operation Procedure (E 1). The animals used in the test were less than 24 hours old were obtained from parent animals aged between 2-4 weeks. Daphnia were originally obtained from Wil Research (Formerly NOTOX B.V.) Hambakenwetering 7, 5231 DD ‘s-Hertogenbosch. The sensitivity of the Daphnia was checked by performing an inhibition test with a reference compound (potassium dichromate) twice a year. The sensitivity was tested for compliance with the relevant study guideline.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
The test was inspected at 0, 24 and 48 hours.
Hardness:
Test medium: Containing per liter of de-ionized water: 200 mg of CaCl2•2H2O, 180 mg of MgSO4•7H2O, 100 mg of NaHCO3 and 20 mg of KHCO3
Test temperature:
22 ± 1°C.
pH:
Test medium: ca. pH 8.2
Conductivity:
Test medium: 550-650 s/cm
Nominal and measured concentrations:
Nominal test concentrations: 4.0, 8.8, 19.4, 42.5, and 93.7 mg/L
Geometric mean concentrations: 2.2, 2.7, 2.4, 13.0 and 56.4 mg/L
Details on test conditions:
Principle of the test:
Juvenile daphnids (<24h old) were exposed to a series of concentrations of the test substance in the test medium, Dutch Standard Water. Under otherwise identical test conditions the effects on the mobility of Daphnia magna exposed to the test substance relation to a control without test substance were recorded over a period of 48 hours.

Test guidelines, modifications and deviations:
The Daphnia magna immobilization test was performed in accordance with OECD Guideline for testing of chemicals number 202 (Ref 1) and Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Annex V, PART C: METHODS FOR THE DETERMINATION OF ECOTOXICITY, C.2:, Daphnia Acute Immobilization test“, Official Journal of the European Union, L 142, Volume 51, 31 May 2008, with no modifications or deviations.

Chemicals:
All other reagents used were of reagent grade quality.

De-ionised water:
The de-ionised water used in the study contained less than 10 μg/L of copper (not measured under GLP), with a conductivity of less than 5 μS/cm and less than 2.0 mg/L NPOC-content.

Test vessels:
As test vessels 50 ml glass beakers were used, containing 50 ml of test solution. All vessels were covered by glass plates during the test.

Test conditions:
The test was carried out in a temperature-controlled room. The test temperature was set at 22°C and the actual temperature should remain constant within ± 1°C. The light regime was 16 h of ambient light per day, provided by fluorescent tubes.

Test medium:
The test medium was Dutch Standard Water (DSW), having a pH of approximately 8.2, and a conductivity between 550 and 650 s/cm, containing per liter of de-ionized water: 200 mg of CaCl2•2H2O, 180 mg of MgSO4•7H2O, 100 mg of NaHCO3 and 20 mg of KHCO3. The guideline criteria requires the CaCO3 content to be between 10 and 250 mg/L (approximately 1-14 ºdH). Water hardness parameters are measured monthly in the DSW system using the appropriate Dr Lange test kit. These are validated by analyzing a CaCl2 Standard. The dilution water was saturated with oxygen before the start of the test.

Other apparatus:
Apparatus was used according to the relevant Standard Operation Procedure as detailed in the study plan. The dissolved oxygen concentrations and conductivity were determined electrochemically using an oxygen electrode / conductivity electrode and meter. The pH was determined with a pH meter. The temperature was measured with a temperature sensor and recorder. Total hardness was measured using Dr Lange test kits and checked for accuracy with appropriate standard solutions.

Test conditions:
The dissolved oxygen and pH was determined before the start and at the end of the test at all test concentrations and in the control. During the test the temperature in the test room was measured continuously in an additional test vessel filled with de-ionized water only under identical conditions as the actual test vessels. The hardness in the batch of water used for testing was checked with reference to the most recent logbook entry.

Test procedures:
The test was performed as a static test for 48 hours. 20 animals divided into 4 batches of 5 animals were tested at each of 5 test concentrations and in the control. Those animals which are not able to swim within 15 seconds after gentle agitation of the test vessel are considered to be immobile. The number of animals being trapped at the surface was determined. These animals were not regarded as immobile and were made to re-submerge. In summary the daphnids were randomly placed in the test fluids and the test vessels were placed in a random manner within each group. The test vessels were not aerated during the test and the animals were not fed. The test was inspected at 0, 24 and 48 hours.

Evaluation of data:
The Lowest Observed Effect Concentration (LOEC) was determined by comparison of the growth at each concentration and the control using threshold values from the Dunnetts test (Dunnetts, 1955). The No Observed Effect Concentration (NOEC) was derived from the results as the first concentration below the LOEC value, where growth shows no significant mobility inhibition relative to the control values. Confidence limits were computed on the basis of Fieller’s theorem if possible (Zerbe, 1978). EC50 values were calculated if possible using the Trimmed Spearman Karber method. All computations were performed using Toxcalc software (Toxcalc, 1994).

Sampling:
The samples were analyzed immediately after sampling.

Analyses:
Physico-chemical parameters and purity of algae
The pH of all samples and controls were measured at the beginning (t=0h) and at the end (t=72h) of the test. The temperature in the culturing apparatus was continuously measured and read out at the end of the test. The light intensity was measured at the beginning and at the end of the test. At the end of the test five random samples were microscopically checked for purity of the algal culture.

Chemical analyses
The chemical analyses were performed on all samples taken.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
>= 56.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
13 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
56.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Water quality
The pH & O2 measurements met study plan requirements.
The temperature varied from 20.6 to 21.4 °C during the test and therefore met the study plan requirements.
Conductivity was 611 μS/cm was 12.2 º dH or 217 mg/L as calcium carbonate during the periodic check of the DSW system in the month of the study. Water quality parameters from the study plan or guideline were therefore also met.

Analytical results:
The concentration of the test substance present in the test vessels was quantified using by HPLC.
The concentrations measured at the start and the end of the test were not within 80 to 120% of the nominal concentrations. Therefore, geometric measured mean concentrations were used for endpoint calculations. Where recovery was
Statistical evaluation of effects:
The results reported are based on geometric measured mean substance concentrations. All reported concentrations refer to the test substance as received and are thus not corrected for active ingredient content.
The NOEC of the test substance was determined as 13 mg/L. No EC50 was reached due to the unexpectedly low sensitivity of the test substance to Daphnia magna. The EC50 may be expressed as >56.4 mg/L, which was the geometric mean of measurements at the highest test concentration.
Results with reference substance (positive control):
The EC50 value of the reference compound, potassium dichromate, was in the range of 0.25-2.0 mg/L (Documented as part of GLP laboratory maintenance).

Quality criteria

The following quality criteria have been met in the present study:

· Immobilization in the control did not exceed 10%

· Oxygen concentration did not fall below 3 mg/L at any point during the study

· The EC50 value of the reference compound, potassium dichromate, was in the range of 0.25-2.0 mg/L (Documented as part of GLP laboratory maintenance).

· No daphnids were trapped on the surface for any length of time

The following quality criterion was not met in the present study:

· The measured concentration of the main component of the test chemical did not remain stable during the study. The test substance was however quantifiable at the end of the study and geometric mean concentrations were therefore calculated for expression of the endpoints as is acceptable in the study guideline.

Table 1- Biological observations

Nominal Concentration mg/L

 

0 hours

24 hours

48 hours

Control

I

5

5

5 (1F)

 

II

5

5

5

 

III

5

5

5

 

IV

5

5

5

Total

 

20

20

20

4

I

5

5

5 (1F)

 

II

5

5

5

 

III

5

5

5

 

IV

5

5

5

Total

 

20

20

20

8.8

I

5

5

5

 

II

5

5

5 (1F)

 

III

5

5

5

 

IV

5

5

5

Total

 

20

20

20

19.4

I

5

5

5

 

II

5

5

5

 

III

5

5

5

 

IV

5

5

5

Total

 

20

20

20

42.5

I

5

5

5

 

II

5

5

5

 

III

5

5

5

 

IV

5

5

5

Total

 

20

20

20

93.7

I

5

5

4

 

II

5

3

8

 

III

5

5

3

 

IV

5

5

4

Total

 

20

18

13

F=Floating test organism (Reintroduced to water phase)

Table 2 – pH Measurements

Nominal test concentration (mg/L)

Time (hours)

0h

48h

Control

8.0

8.2

4

8.0

7.8

8.8

8.0

7.8

19.4

8.0

7.9

42.5

8.0

7.8

93.7

8.0

7.8

Table 3 –Oxygen Measurements

Nominal test concentration (mg/L)

Time (hours)

0h

48h

Control

8.8

8.8

4

8.8

8.8

8.8

8.8

8.8

19.4

8.8

8.9

42.5

8.8

8.8

93.7

8.8

8.7

Validity criteria fulfilled:
yes
Conclusions:
All of the biological and analytical quality criteria were met and the study data can be considered to be an accurate representation of the effects of the test material to Daphnia magna in the chosen concentration range. The effects of the test material to Daphnia magna were however underestimated and the chosen test range was not optimal. Due to the algae endpoint in T14009 AL being many times (Approximately 100 x) more sensitive, this study need not be repeated as the algae value will drive
classification and risk assessment.
Executive summary:

In order to predict the effects of the test substance in an aquatic environment, an acute immobilization test to Daphnia magna was conducted in accordance with OECD test guidelines and with the OECD

Principles of Good Laboratory Practice.

The toxicity of the test chemical to juvenile daphnids was determined in a static system over an exposure period of 48 hours. Nominal concentrations of 4.0, 8.8, 19.4, 42.5, and 93.7 mg/L were tested including the required control group.

A NOEC of 13 mg/L and a LOEC of 56.4 mg/L were determined. The EC50 (48h) may be expressed as >56.4 mg/L as 50 % effect was not reached at the highest concentration.

Geometric measured mean concentrations were used for endpoint determination due to the recovery of the main component falling below 80% of the nominal concentrations.

The following quality criteria have been met in the present study:

· Immobilization in the control did not exceed 10%

· Oxygen concentration did not fall below 3 mg/L at any point during the study

· The EC50 value of the reference compound, potassium dichromate, was in the range of 0.25-2.0 mg/L (Documented as part of GLP laboratory maintenance).

· No daphnids were irreversibly trapped on the surface for any length of time

· Chemical analysis quality criteria were met and although the test material was not stable in the test system quantification could still be made in the region of effect allowing geometric mean

concentrations to be used.

· The maximum variation in pH over the full test period was 0.8 pH units which meets the guideline pH variation requirements

The aim of the study was to determine a concentration at which 50% effect occurs in the test organism. Due to low sensitivity of the test species this was not achieved. However due to the high sensitivity of

the algae endpoint the data generated in this study is sufficient to indicate that Daphnia magna toxicity is not the driver of classification and labeling or risk assessment for this substance.

Description of key information

A valid acute Daphnia study according to OECD 202 performed under GLP is available.

The EC50(48h) may be expressed as >56.4 mg/L as 50 % effect was not reached at the highest concentration.

Geometric measured mean concentrations were used for endpoint determination due to the recovery of the main component falling below 80% of the nominal concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
56.4 mg/L

Additional information