Registration Dossier

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 May 2011 - 1 June 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items and those which degrade rapidly in the test system, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a saturated solution of the test item in cases where the test item is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (50 mg/L) of test item in reconstituted water for a period of 24 hours prior to removing any undissolved test item present by filtration (0.2 µm Sartorius Sartopore, first approximate 1 litre discarded in order to pre-condition the filter) to give a saturated solution of the test item hydrolysis product.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification: Experimental ALC20110128 Exp1
Description: off white powder
Storage conditions: room temperature in the dark

The integrity of supplied data relating to the identity, purity and stability of the test item was the responsibility of the Sponsor.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Verification of test concentrations:
Water samples were taken from the control (replicates R1 - R4 pooled) and the 100 % v/v saturated solution test group (replicates R1 - R2 and R3 - R4 and pooled) at 0 and 48 hours for quantitative analysis. The 0 h samples were stored at approximately -20 °C prior to analysis.
Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
Pre-Study Media Preparation Trials:
Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Based on this information the test item 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 media preparation trials were conducted in order to determine the solubility of the test item under test conditions.

1. Saturated Solution Preparation:
An amount of test item (550 mg) was dispersed in 11 litres of deionised reverse osmosis water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:
-Centrifugation at 10000 x gravity for 30 minutes
-Centrifugation at 40000 x gravity for 30 minutes
-Filtration through a 0.2 µm Sartorius Sartopore filter (approximately 1 litre discarded in order to pre-condition the filter)
-Filtration through a 0.2 µm Sartorius Sartopore filter (approximately 2 litres discarded in order to pre-condition the filter)

2. Results:
Test Sample Concentration Found (mg/L)
Centrifuged 10000 x gravity 10.8
Centrifuged 40000 x gravity 12.2
Filtered - 1 litre discarded Filtered - 2 litres discarded
LOQ = Limit of quantitation

3. Discussion:
Visual observations made on the centrifuged test samples prior to analysis indicated that undissolved hydrolysis product remained which would account for the high results obtained. As such the use of centrifugation was considered inappropriate for this test item.
Based on this information the test item was prepared using a saturated solution method of preparation at an initial loading rate of 50 mg parent test item/L, stirred for a period of 24 hours prior to the removal of any undissolved hydrolysis product by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 1 litre discarded) to give a 100 n% v/v saturated solution.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Source: In-house laboratory cultures.
- Age at study initiation (mean and range, SD): 1st instar. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.
- Method of breeding: Culture conditions ensured that reproduction was by parthenogenesis.
- Feeding during test: No
The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

ACCLIMATION
- Type and amount of food: Each culture was fed with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin flake food suspension.
- Feeding frequency: daily

Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at 21 °C - 22 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h

Test conditions

Hardness:
Approximate total hardness of 250 mg/mL as CaCO3
Test temperature:
21 - 22 °C
pH:
7.8 - 8.4
Dissolved oxygen:
8.7 - 9.3 mg O2/L

The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100 %. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed in the control group.
Nominal and measured concentrations:
Nominal Concentrations: 0, 0.10, 1.0, 10 and 100 % v/v Saturated Solutions.

Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained which was determined to be 0.13 mg/L. This does not infer that no hydrolysis product was in solution, just that what was soluble was at a concentration of less than the LOQ.
Details on test conditions:
TEST WATER
Reconstituted water was used for both the range-finding and definitive tests.
-Stock Solutions
CaCI2.2H2O 11.76 g/L
MgSO4.7H2O 4.93 g/L
NaHCO3 2.59 g/L
KCI 0.23 g/L
-Preparation
An aliquot (25 mL) of each of solutions 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 HCI and was aerated until the dissolved oxygen concentration was approximately air-saturation value.

ELENDT M7 MEDIUM
Solution Concentration of stock solution (mg/L)
H3BO3 57190
MnCI2.4H2O 7210
LiCI 6120
RbCI 1420
SrCI2.6H20 3040
NaBr 320
Na2MoO4.2H2O 1260
CuCI2.2H2O 335
ZnCI2 260
CoCI2.6H2O 200
KI 65
Na2SeO3 43.8
NH4VO3 11.5
Na2EDTA.2H2O 5000
FeSO4.7H2O 1991
An aliquot (dependant on the volume of medium required) of each stock solution was added to a final volume of deionised reverse osmosis water to give stock solution A and stored at approximately 21 °C.

Macro Nutrient Stock Solutions
Solution Concentration of stock solution (g/L)
CaCI2.2H2O 293.80
NaHCO3 64.80
MgSO4.7H2O 246.60
Na2SiO3.9H2O 50.00
KCI 58.00
NaNO3 2.74
K2HPO4 1.84
KH2PO4 1.43

Vitamin Nutrients
Solution Concentration of stock solution (mg/L)
Thiamine hydrochloride 750
Vitamin B12 100
D (+) biotin (vitamin H) 75

The final medium was prepared by adding an aliquot of stock solution A along with aliquots of each individual Macro Nutrient Stock Solution and an aliquot of the vitamin nutrient to the required amount (final volume) of deionised reverse osmosis water.
The pH of the prepared media was 8.0 ± 0.2 and stored at approximately 21 °C.

PROCEDURE
Range-finding test:
The test concentration to be used in the definitive test was determined by a preliminary range-finding test. In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 % v/v saturated solution.
An amount of test item (550 mg) was dispersed in 11 litres of reconstituted water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved hydrolysis product was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 1 litre discarded in order to pre-condition the filter) to give a 100 % v/v saturated solution. Serial dilutions were prepared in reconstituted water from this saturated solution to give further test concentrations of 10, 1.0 and 0.10 % v/v saturated solution. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at approximately 20 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 250 mL test and control vessel contained 200 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised Daphnia magna were recorded.
The control group was maintained under identical conditions but not exposed to the test item.

Definitive test:
Based on the result of the pre-study media preparation trial and range-finding test a "limit test" was conducted at a concentration of 100 % v/v saturated solution to confirm that at the highest attainable concentration no immobilisation was observed.

Experimental preparation:
For the purpose of the definitive test the test item was prepared as a saturated solution.
An amount of test item (550 mg) was dispersed in 11 litres of reconstituted water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved hydrolysis product was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 1 litre discarded in order to pre-condition the filter) to give a 100 % v/v saturated solution.
The concentration of the hydrolysis product in the test preparation was verified by chemical analysis at 0 and 48 hours.

Exposure conditions:
As in the range-finding test 250 ml glass jars containing approximately 200 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 21 °C to 22 °C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were not renewed during the exposure period. 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.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrations
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
Details on results:
Range-finding Test:
No immobilisation was observed at all test concentrations.
Based on this information, a single test concentration of four replicates, of 100 % v/v saturated solution was selected for the definitive test.

Definitive Test
Immobilisation data:
There was no immobilisation in 20 daphnids exposed to a test concentration of 100 % v/v saturated solution for a period of 48 hours.
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.

Observations on test item solubility:
The 100 % v/v saturated solution test concentration was observed to be a clear, colourless solution throughout the duration of the test.
Results with reference substance (positive control):
The results from the positive control were within the normal range for this test item.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48 hour EC50 of greater than 100 % v/v saturated solution. The No Observed Effect Concentration was 100 % v/v saturated solution.
The study showed no toxic effects at saturation.
Executive summary:

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48 hour EC50 of greater than 100 % v/v saturated solution. The No Observed Effect Concentration was 100 % v/v saturated solution.

The study showed no toxic effects at saturation.

The study was conducted in accordance with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.