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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:
experimental study
Adequacy of study:
key study
Study period:
May-July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction 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 (migrated information):
None.
Analytical monitoring:
yes
Details on sampling:
Test concentrations: control ; 0.1 ; 0.32 ; 1 ; 3.2 ; 10 mg/L

Water samples were taken from the control and each surviving test group for quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 5, 8, 12, 15, 19 and 20 (from the bulk test preparations) and of the expired test preparations on Days 1, 6, 9, 13, 16, 20 and 21 (replicates pooled). Duplicate samples were taken and stored frozen for further analysis if necessary. Due to a technical error, no Day 0 results are available.

The fresh samples were analysed immediately for test item concentration determination.
Vehicle:
no
Details on test solutions:
A nominal amount of test item (50 mg) was dissolved in test water with the aid of ultrasonication for approximately 15 minutes and the volume adjusted to 5 liters to give the test concentration of 10 mg/L. Dilutions were made from the 10 mg/L test concentration to give the remaining test concentrations of 0.10, 0.32, 1.0 and 3.2 mg/L. Due to the light sensitive nature of the test item, all preparation was carried out under laboratory safety lighting. Prior to use, the test item and the test water used to prepare the 10 mg/L test concentration were heated to approximately 37°C. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minutes dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. 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. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
None.
Hardness:
The water hardness was observed to be in the range 226 to 254 mg/L as CaCO3 in the control and the highest surviving test group throughout the test.
Test temperature:
19-21°C (min-max)
pH:
7.1-8.0 (min-max)
Dissolved oxygen:
6.0-9.0 mg/L (min-max)
Salinity:
Close to 0 mg/L
Nominal and measured concentrations:
Nominal concentrations: 0.1 ; 0.32 ; 1 ; 3.2 ; 10 mg/L
Calculated concentration (time weighted average): 0.032 ; 0.08 ; 0.15 ; 2.9 ; 4.3 mg/L
Details on test conditions:
For each concentration a single daphnid was placed in approximately 150 mL of the test preparation in 150 mL glass vessels which were sealed to prevent any losses through evaporation and any potential losses through volatilization. For each test and control group ten replicate test vessels were prepared. The test vessels were maintained in a temperature controlled room at approximately 20 °C in darkness for 21 days. The test vessels were not aerated. The diluent water only was aerated prior to use.
The control group was maintained under identical conditions but not exposed to the test item. The test preparations were renewed daily. 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 using before being discarded.
Each daphnid received approximately 5 to 10 µL of an algal suspension (Desmodesmus subspicatus) and approximately 10 to 30 µL of Tetramin® flake food suspension daily. 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.
Reference substance (positive control):
not specified
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
3.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
3.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
> 4.3 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth
Details on results:
Lethal Effects on the Parental Generation (P1)
Mortality (immobilization) occurred predominantly at the highest test concentration of 10 mg/L resulting in 40% mortality at the end of the study.Mortality was also observed at the test concentrations of 0.10, 0.32 and 3.2 mg/L. Analysis of the data using the Fishers Exact Test showed that mortalities in the 10 mg/L test group were not significant, however, upon inspection of the data it is considered that there was a significant effect on survival in the 10 mg/L test group. EC50 > 4.3 mg/L and EC10 = 3.7 mg/L

Sub-lethal Effects on the Parental Generation (P1)
There was no significant effect on the size or color of the daphnids at each concentration. After 21 days the length of each surviving adult was determined. The results showed that there were no statistically significant differences (P= 0.05) between the control and each test group in terms of length of the daphnids after 21 days exposure to the test item.

Effects on Reproduction
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. This was true for all versions of analysis (i.e. total young production, young production excluding any inadvertent mortalities, and young production excluding all immobilized adults). EC50 > 4.3 mg/L and EC10 = 3.7 mg/L
Results with reference substance (positive control):
None.
Reported statistics and error estimates:
The EC50 and EC10 (immobilization) values on Day 21 were calculated by the Linear Interpolation method using the ToxCalc computer software package (ToxCalc 1999).
The observed mortalities in the parental (P1) generation of the 0.10, 0.32, 3.2 and 10 mg/L test groups were compared to the control group using the Fishers Exact Method using the ToxCalc computer software package (ToxCalc 1999).
The EC10 (reproduction) value on Day 21 were calculated by the Linear Interpolation method using the ToxCalc computer software package (ToxCalc 1999). The EC50 (reproduction) value after 21 days was estimated by inspection of the data.
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). Daphnia length data, determined for the surviving daphnids on termination of the test from 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 Dunnett’s multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

DUNNETT, C W (1955) A Multiple Comparison Procedure for Comparing Several Treatments With a Control. J Am Stat Assoc 50, 1096-1121.
ToxCalc Version 5.0.23C (1999), Tidepool Scientific Software, McKinleyville, CA 95519, USA.
WILLIAMS, D A (1971) A Test for Differences Between Treatment Means When Several Dose Levels are Compared With a Zero Dose Control. Biometrics 27, 103-117.
Validity criteria fulfilled:
yes
Conclusions:
A GLP OECD 211 study was performed to test the toxicity of undecylenic acid to daphnia magna reproduction. No effect was observed on neonate production or parental growth. An EC10 of 3.7 mg/L was calculated for parental immobilization.
Executive summary:

A GLP OECD 211 study was performed to test the toxicity of undecylenic acid to daphnia magna reproduction. Concentrations of 0.1 ; 0.32 ; 1 ; 3.2 and 10 mg/L were tested. Since the test item was unstable in the medium, time weighted average concentrations were used for statistical purposes. No adverse effect was observed on neonate production or parental growth. An EC10 of 3.7 mg/L was calculated for parental mortality (immobilization).

Description of key information

The chronic toxicity of the test item 10-UNDECENOIC ACID to Daphnia magna was assessed according to the OECD guideline 211 with semi-static conditions (Harris, 2014). Daphnids were exposed to a range of concentrations of 10-UNDECENOIC ACID dissolved in dilution water. The chronic effect measured during the assay was the inhibition of reproduction (neonate production, parental growth and parental immobilization) after a 21 day-period. No effect were observed on parental growth. A 21d-EC10 of 3.7 mg/L was found for both parental immobilization and neonate production (time weighted average concentration).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
3.7 mg/L

Additional information