Registration Dossier

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-05-07 until 2014-06-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Qualifier:
according to guideline
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material: Refer to respective sections of IUCLID fiele for details.
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Based on the data supplied by the Sponsor the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.
- Sampling method: The concentration and stability of the test item in the test preparations were verified by chemical analysis on Days 0, 5, 12 and 20 (fresh media) and on Days 2, 5, 7, 14 and 21 (old media). The adult daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted before being discarded.
- Sample storage conditions before analysis: The samples were stored frozen prior to analysis
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item concentration in the test samples was determined by high performance liquid chromatography with mass spectrometry (HPLC-MS) using an external standard. The test item gave a chromatographic profile consisting of a single peak. The reconstituted water (Elendt M7 medium) used for the definitive test was the same as that used to maintain the stock animals.
- Eluate:0.1% (v/v) formic acid in acetonitrile:water (50: 50, v/v)
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Not relevant
- Justification for species other than prescribed by test guideline: Not relevant
- Source: In-house laboratory cultures.
- Age of parental stock (mean and range, SD): 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.
- Feeding during test: Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.

ACCLIMATION
- Acclimation period: Not reported

QUARANTINE (wild caught): Not relevant

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Not relevant
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Post exposure observation period:
None
Hardness:
Range of ca. 226 to 260 mg/L (as CaCO3)
Test temperature:
Approximately 20°C. The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
pH:
pH was in range of ca. 7.7 to 8.0. The pH was measured using a Hach HQ30d Flexi handheld meter.
Dissolved oxygen:
Range of ca. 8.8 to 9.1 mg O2/L.The dissolved oxygen concentration was measured using a Hach HQ30d Flexi handheld meter.
Salinity:
Not detected
Nominal and measured concentrations:
The concentration and stability of the test item in the test preparations were verified by chemical analysis on Days 0, 5, 12 and 20 (fresh media) and on Days 2, 5, 7, 14 and 21 (old media).
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL glass vessels
- Type (delete if not applicable): closed (covered with a plastic lid to reduce evaporation)
- Material, size, headspace, fill volume: glass, 100 mL fill volume
- Aeration: The diluent water only was aerated prior to use. The test vessels were not aerated.
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate): Test preparations were renewed 3 times per week on Days 0, 2, 5, 7, 9, 12, 14, 16 and 20
- No. of organisms per vessel: single daphnid per vessel
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10 vessels. The control group was maintained under identical conditions but not exposed to the test item.
- No. of vessels per vehicle control (replicates): Not relevant

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water - Elendt M7 Medium
Final concentrations of ingredients (in mg/L):
H3BO3: 0.715,
MnCl2.4H20: 0.090,
LiCl: 0.077,
RbCl: 0.018,
SrCl2.6H20: 0.038,
NaBr: 0.004,
Na2Mo04.2H20: 0.016,
CuCl2.2H20: 0.004,
ZnCl2: 0.013,
CoCl2.6H20: 0.010,
KI: 0.0033,
Na2Se03: 0.0022,
NH4VO3: 0.00058,
Na2EDTA.2H20: 0.625,
FeS04.7H20: 0.249,
CaCl2.2H20: 293.8,
NaHC03: 64.8,
MgS04.7H20: 123.3,
Na2Si03.9H20: 10,
KCl: 5.8,
NaN03: 0.274,
K2HP04: 0.184,
KH2P04: 0.143,
Thiamine hydrochloride: 0.075,
Cyanocobalamine (vitamin B12): 0.0010,
D(+) biotin (vitamin H): 0.00075.

OTHER TEST CONDITIONS
- Adjustment of pH: Not adjusted
- Photoperiod: photoperiod of 16 hours light (710 to 786 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days.
- Light intensity: 710 to 786 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobilization:
Those organisms which are not able to swim within 15 seconds after gentle agitation of the test container are considered to be immobile.
Reproduction Rate:
Expressed as:
1. The total number of living offspring produced.
2. The total number of living offspring produced per parent daphnia surviving until the end of the test.
3. The total number of living offspring produced at the end of the test per parent daphnia at the start of the test excluding from the analysis parental accidental and/or inadvertent mortality.

VEHICLE CONTROL PERFORMED: Not relevant

RANGE-FINDING STUDY: Not relevant
Reference substance (positive control):
not required
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: adult growth
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: adult growth
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: adult growth
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: adult growth
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
other: MATC (Maximum Acceptable Toxicant Concentration)
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Result calculated from lowest calculated NOEC and LOEC
Remarks on result:
other: Result calculated from lowest calculated NOEC and LOEC
Details on results:
- Mortality of parent animals:
Single immobilized daphnids were observed in the control and the 3.2, 32 and 100 mg/L test groups. In addition, 20% immobilization was observed in the 10 mg/L test group. This immobilization was considered not to impact the test as this did not exceed 20% and also did not follow a concentration-dependent pattern. No immobilization occurred at the 1.0 mg/L test concentration throughout the test.
- No. of offspring produced per day per female:
Analysis of the data obtained on Day 21 showed that the numbers of live young produced per adult by the control group were not significantly different (P>0.05) from each test group.
- Body length and weight of parent animals: No effects observed
- Type and number of morphological abnormalities: No effects observed
- Type and number of behavioural abnormalities: No effects observed
- Number of males and females (parental): No effects observed
- Time to first brood release or time to hatch: Young were first produced in the control group on Day 8 of the test.
There were no unhatched eggs or dead young observed during the test.
- Other biological observations: None
- Effect concentrations exceeding solubility of substance in test medium: None
Results with reference substance (positive control):
No positive control implemented.
Reported statistics and error estimates:
For the estimation of the "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) the numbers of live young produced per adult over the duration of the test for the control and each test group were compared using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and the Williams test for differences between treatment means when several dose levels are compared with a zero dose control (Williams 1971) using the SAS computer software package (SAS 1999 - 2001). Results from the control and each test group daphnia length data, determined for the surviving daphnids on termination of the test, were compared using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) using the SAS computer software package (SAS 1999 -2001). The NOEC and LOEC for parental immobilization were determined by Fisher's Exact Test using the ToxCalc computer software package (ToxCalc 1999).

The detection system was found to have acceptable correlation to the fit of a quadratic equation. The analytical procedure was found to have acceptable recoveries of test item in test medium. The method of analysis was validated and proven to be suitable for use. Due to the high sensitivity of the analytical method (LOQ = 0.00051 mg/L), measurable levels were detected in nearly all the test controls although all blanks and synthetic controls were less than the LOQ.

Analysis of the 100 mg/L test preparation on Days 0, 5, 12 and 20 (fresh media) and on Days 2, 5, 7, 14 and 21 (old media) showed measured test concentrations to range from 80% to 94% of nominal value and so the results are based on nominal test concentrations only.

Validity criteria fulfilled:
yes
Conclusions:
No toxic effects of the test compound towards Daphnia magna were observed, even at the highest concentration tested (100 mg/L).
Executive summary:

A study was performed to assess the chronic toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2012) No 211, "Daphnia magna Reproduction Test" referenced as Method C.20 of Commission Regulation (EC) No. 440/2008 and the US EPA Draft Ecological Effects Test Guidelines OCSPP 850.1300 "Daphnid Chronic Toxicity Test".

Based on data supplied by the Sponsor, Daphnia magna were exposed (10 replicates of a single daphnid per group) to an aqueous solution of the test item over a range of test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L for a period of 21 days. The test solutions were renewed 3 times per week. The numbers of live and dead adult daphnia and young daphnids (live and dead) were determined daily. The daphnia were fed daily with a mixture of algal suspension and Tetramin® flake food suspension.

Results:

Analysis of the 100 mg/L test preparation on Days 0, 5, 12 and 20 (fresh media) and on Days 2, 5, 7, 14 and 21 (old media) showed measured test concentrations to range from 80% to 94% of nominal value and so the results are based on nominal test concentrations only.

The 21-Day EC50 (immobilization) value, based on nominal test concentrations for the parental Daphnia generation (Pi) was estimated to be greater than 100 mg/L. The 21-Day EC50 (adult growth) based on nominal test concentrations was greater than 100 mg/L. The 21-Day EC50 (reproduction) based on nominal test concentrations was greater than 100 mg/L. The "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) based on nominal test concentrations were 100 mg/L and greater than 100 mg/L for parental immobilization, adult growth and reproduction, respectively. The "Maximum Acceptable Toxicant Concentration" (MATC) was calculated to be 100 mg/L.

,

Description of key information

A study was performed to assess the chronic toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2012) No 211, "Daphnia magna Reproduction Test" referenced as Method C.20 of Commission Regulation (EC) No. 440/2008 and the US EPA Draft Ecological Effects Test Guidelines OCSPP 850.1300 "Daphnid Chronic Toxicity Test".

The 21-Day EC50 (immobilization) value, based on nominal test concentrations for the parental Daphnia generation (Pi) is greater than 100 mg/L. The 21-Day EC50 (adult growth) based on nominal test concentrations is greater than 100 mg/L. The 21 -Day EC50 (reproduction) based on nominal test concentrations is also >100 mg/L. The "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) based on nominal test concentrations are 100 mg/L and greater than 100 mg/L for parental immobilization, adult growth and reproduction, respectively. The "Maximum Acceptable Toxicant Concentration" (MATC) amounts to 100 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information