Registration Dossier

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

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:
23-5-2012- 4-10-2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study to GLP meeting biological and analytical quality criteria. Analysis condcted as an additional stability test (by study plan amendment) under identical conditions due to failure of original analytical method. Mainaining exposure to parent was not possible. Test solution was most likely primarily degradation components that were not quantified. Valid but with these restrictions.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
See below
Principles of method if other than guideline:
For stability reasons the test chemical is mixed with Isododecane. Testing without the presence of Isododecane is not feasible for stability and safety reasons. The marketed product was therefore considered a multi component substance containing a mixture of individual substances with different solubility and physical chemical properties. The test chemical was therefore be tested as such, using the water accommodated fraction method (WAF). Furthermore the test material is instable and hydrolizes and thermally decomposes relitively rapidly to multiple degradation products. The WAF method therefore allows the hazard of the resulting test material as a whole to be determined.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Sampling was intended for the start and for the end of the test. However the tests running in parallel had indicated that the test substance was no longer detectable after 24 hours. For this reason only two samples were analyzed. 0 (representative of the start of the test after stirring period) and 24 hours representative of the first 24 hours of the test. After which the test solutions were refreshed. Hence the test consisted of 2 x 24 h. Due to the original analysis method not meeting the required quality criteria analysis was repeated under indentical conditions and concentrations but independant of the biological part of the test.
Details on test solutions:
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 (Standard Operation Procedure O1) . The guideline criteria requires the CaCO3 content to be between 10 and 250 mg/L (approximately 1-14 ºdH). Hardness in the test water was measured once at the start of the study using the appropriate Dr Lange test kit. This was validated by analyzing a CaCl2 Standard. The dilution was saturated with oxygen before the start of the test. This is done by thorough aeration for at least 30 minutes before use.

General test principles and procedures
The test was performed semi static test for 48 hours. 20 animals divided into 4 batches of 5 animals were tested in the control and at each test concentration. Those animals which are not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobile. The number of animals trapped at the surface was also considered and recorded if observed. 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. Due to stability indications from preliminary work the solutions were replaced after 24 hours with the original WAF solutions that had been restarted. Animals were carefully transferred in all cases to test replicates using a glass pipette.

Preparation of solutions
The test chemical is poorly soluble and potentially prone to hydrolysis and thermal degradation. For this reason a preparation method that allows the test chemical and potentially any hydrolytic or thermal degradation products the possibility to dissolve up to their maximum solubility limit in the test media was chosen. A WAF approach was therefore used for this test. WAF vessels were not diluted from a stock but were made separately.

All WAF solutions were prepared for the test concentrations by adding an accurate amount of the test substance to the test media and allowing it equilibrate under slow agitation over 72 hours in a sealed vessel. It was then considered loaded with the test substance (at maximum achievable solubility) and breakdown products. Analytical measurments were conducted in support of this.

After the stirring period the agitation for all replicates was ceased and the solutions were left to stand for 1 hour. Due to the chance of dispersion formation solutions were extracted carefully from the middle of the WAF vessel and centrifuged at 8000 RPM for 10 minutes. The resulting solutions with the exception of the last few millimeters of fluid in the centrifuge tube were used directly for testing. The WAF solutions were restarted and resealed in order to be used for the solution refreshment.

Due to the expected degradation of the dissolved fraction of parent material the test vessels were replenished after 24 hours (with original WAF solutions shown to be stable in the water solubility test) to ensure a worst case scenario with regards to the parent. Identical procedures to those described were followed after 24 hours and the animals carefully transferred.

Test organisms (species):
Daphnia magna
Details on test organisms:
The test animals were taken from a Daphnia magna stock, cultured in conformity with the relevant Stan¬dard Operation Procedure. 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 NOTOX B.V. Hambakenwetering 7 5231 DD ‘s-Hertogenbosch P.O. Box 3476 5203 DL ‘s-Hertogenbosch the Netherlands.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
ºdH of 14.9 which is equivalent to approximately 266 mg/L as CaCO3.
Test temperature:
20.8 to 21.2 °C .
pH:
pH remained stable in the test in all concentrations.
Dissolved oxygen:
The oxygen concentration did not fall below 8.7 mg/L at any point during the test.
Salinity:
Not Measured.
Nominal and measured concentrations:
Control, 10.0, 22.0, 48.4, 106.4, and 234.3 mg/L
Measured concentration of parent concentration in parallel analytical study shown in table below .
Details on test conditions:
TEST SYSTEM
- Test vessel:50 ml Glass Beaker
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 50ml Approx
- Aeration: All medium before use

- Renewal rate of test solution (frequency/flow rate):After 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates):4
- No. of vessels per control (replicates):4


OTHER TEST CONDITIONS

- Photoperiod: 16 h of ambient light per day, provided by fluorescent tubes.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobilisation

TEST CONCENTRATIONS
- Spacing factor for test concentrations:2.2
- Range finding study Four tests with different substance preparation methods carried out. Attached as supporting study.
- Results used to determine the conditions for the definitive study: Attached as supporting study.
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 234.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(21.1µg/L measured initial parent)
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
234.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
No effects were observed on the test organisms regardless of loading. An EC50 could not be reached. Due to the instable nature of the parent test substance the solutions were refreshed after 24 hours. The test substance may be concluded as not toxic to daphnia magna at the maximum achievable solubility in test medium and data indicates that degradation products are also not of high concern. Consideration should be made to the fact that only dissolved test material was transferred to the test system to avoid physical entrapment and avoid testing over the limit of water solubility.
Results with reference substance (positive control):
Carried out as part of the GLP maintainance.
Reported statistics and error estimates:
Not carried out no mortality at highest concentration.

Measured test substance concentrations (Stability test without test organisms)

Name

Measured conc.

(mg/L)

Final conc.

(µg/L)

Nominal conc.

(mg/L)

T11032 Recovery DSW I-1 T=0

0.0194

19.4

12.2

T11032 Recovery DSW I-2 T=0

0.0193

19.3

12.2

T11032 Recovery DSW II-1 T=0

0.0213

21.3

48.8

T11032 Recovery DSW II-2 T=0

0.0187

18.7

48.8

T11032 Recovery DSW III-1 T=0

0.0204

20.4

234.8

T11032 Recovery DSW III-2 T=0

0.0218

21.8

234.8

T11032 Recovery DSW I-1 T=24

<LOD

<LOD

12.2

T11032 Recovery DSW I-2 T=24

<LOD

<LOD

12.2

T11032 Recovery DSW II-1 T=24

<LOD

<LOD

48.8

T11032 Recovery DSW II-2 T=24

<LOD

<LOD

48.8

T11032 Recovery DSW III-1 T=24

<LOD

<LOD

234.8

T11032 Recovery DSW III-2 T=24

<LOD

<LOD

234.8

Results show that despite the large increase in the loading of the test material the soluble fraction of the parent component stays relatively stable. Indicating that the test did indeed take place at the maximum achievable concentration of the parent and most likely in the presence of an increasing concentration of degradation products that were not quantified in this study. Results also indicate that the parent component was completely degraded after 24 hours and no longer detectable. For this reason a semi static approach was used to replenish the test substance and ensure a worst case scenario

Validity criteria fulfilled:
yes
Conclusions:
Due to the very high loadings and the relatively constant measurements of parent test material at concentrations very close to the water solubility and bearing in mind the significant number of preliminary tests conducted, the substance can be concluded as non acutely toxic at its solubility limit.
in the test medium and would not require classification for this endpoint. The lack of complete biodegradation does present some grounds for concern regarding the chronic toxicity potential of the degradation products. Adsorption to test vessels may also have taken place but the nature of the test chemical makes correcting for this not feasible.
Executive summary:

Guideline study with modifications due to the difficult nature of the test substance. Certificate of analysis present. GLP accreditation. Analytical investigation conducted separately to test (but using identical concentrations, medium and procedures) due to failed analytical method during the study itself. Extended range finding work conducted under different preparation methods to justify approach in definitive test. See supporting studies. Correction for potential absorbption to glass or lack of exposure due to glass absorbption cannot be completely excluded despite a solution refreshment being carried out.Exposure to more stable more soluble degradation products would however likely have been sufficient. Therefore a klimisch 1 cannot be given. Study reliable with these restrictions.

Description of key information

The substance can be concluded as non acutely toxic at its solubility limit.

Key value for chemical safety assessment

Additional information