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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:
February 07, 2013 - February 17, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100 mg/L
- Sampling method: The saturated solution of the test item and the control were analysed by HPLC-DAD at the start of the exposure intervals (0 h) on days 0, 9, 14 and at the end of the exposure intervals (48 or 72 h) on days 2, 12, 16. For the analyses at the end of the exposure intervals, separate replicates without algae and daphnids were prepared and stored under test conditions until sampling.
Vehicle:
yes
Remarks:
Elendt M4, according too ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
Details on test solutions:
A saturated solution (100 mg/L) was prepared with dilution water one day before start of each exposure interval. The saturated solution ( 100 mg/l test item were weighed out)
was prepared with dilution water one day before the start of each exposure interval at every water renewal day.

Elendt M4, according too ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L as used as vehicle.

The test design was semi-static with a medium renewal 3 times per week, i.e. after 48 and 72 h.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Daphnia magna Straus (Clone 5)
- Source: lnstitut für Wasser-, Boden- und Lufthygiene (WaBolu)
- Breeder: DR.U. NOACK-LABORATORIEN, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany
- Feeding during test: Daily feeding per test vessel with Pseudokirchneriella subcapitata (0.106 - 0.304 mL) and Desmodesmus subspicatus (0.189 - 0.257 mL) suspension was provided as food corresponding to 0.2 mg C per Daphnia and day.

ACCLIMATION
- Acclimation period: At least 2h in dilution water
- Acclimation conditions: same as test
- Type and amount of food: At least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 10^6 cells/mL
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Hardness:
146 - 180 mg/L CaCO3
Test temperature:
20.6 - 22.0°C
pH:
7.67 - 8.08
Dissolved oxygen:
5.08 - 8.72 mg/L
Nominal and measured concentrations:
Nominal concentration: 100 mg/L
Measured Concentration: 16.1 µg/L (geometric mean measured concentration)
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers 100 mL capacity
- Volume of test medium: 50 mL
- Type : open
- Renewal rate of test solution: renewal 3 times per week, i.e. after 48 and 72 hours
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Culture medium Elendt M4, according to ELENDT ( 1990), modified to a total hardness of 160 to 180 mg CaC03/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: once within 7 days

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16/8h light/dark cycle
- Light intensity: Max. 20 µE m-2 s-1

TEST CONCENTRATIONS
- Range finding study: A non GLP preliminary range finding test was carried out at the test facility as acute immobilisation test to Daphnia magna STRAUS. A limit loading rate of 100 mg/L of the test item was tested under static conditions over a period of 48 h and no immobilisation was seen.
Reference substance (positive control):
yes
Remarks:
A reference test was conducted as an acute immobilisation test (acc. to DIN 38412 L11 and AQS P 9/2) under static conditions with a test duration of 24h once per month. Potassium dichromate was used as reference
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
3.43 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: NOEC = solubility limit
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
3.43 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: NOEC = solubility limit
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
The measured concentrations of the test item at the start of the exposure intervals were in the range of< LOQM to 5.35 μg/L. At the end of the exposure intervals (48 or 72 h), the measured concentrations of the test item were in the range of< LOQM to 3.60 μg/L. The measured values below 70 % of the LOQM (5.00 μg/L) were taken into account with half of the LOQM (= 2.50 μg/L). The geometric mean measured concentration of the test item was calculated to be 3.43 μg/L and was used for the assessment of the effect levels.

The average values of living juveniles per parental daphnid were 75 for the control and 76 for the geometric mean measured concentration of 3.43 μg/L after 21 days. The reproductive output of daphnids in the geometric mean measured concentration of 3.43 μg/L was comparable to the reproductive output of daphnids in the control (t-test, p = 0.05).

The coefficient of variation of the number of living offspring produced per parent was 8 % for both the control and the geometric mean measured concentration of 3.43 μg/L.Generally in a well-run test, the coefficient of varation around the mean number of living offspring per parent animal should be <= 25%.

The intrinsic rates of natural increase (IR) of the surviving parental daphnids accounting for generation time and number of offspring were used for calculation of population growth and maintenance. The intrinsic rates of the geometric mean measured concentration of 3.43 μg/L were comparable to those in the control (t-test, p = 0.05).

No adult mortality was observed during the test period of 21 days. In the geometric mean measured concentration of 3.43 μg/L and in the control, all parental daphnids survived until the end of the test after 21 days.

The first brood was released on days 8 and 9 by all parental daphnids of the control and the geometric mean measured concentration of 3.43 μg/L. At the geometric mean measured concentration of 3.43 μg/L and the control, 4 broods were produced by all parental daphnids.

The mean dry body weight and total body lengths of all parental daphnids of the geometric mean measured concentration of 3.43 μg/L and the control were determined at the end of the study. The mean dry body weight of the parental daphnids was 0.92 mg per daphnid for the control and 0.95 mg per daphnid for the geometric mean measured concentration of 3.43 μg/L. The mean body length of the parental daphnids was 5.20 mm per daphnid for the control and 5.05 mm per daphnid for the geometric mean measured concentration of 3.43 μg/L.

No stillborn juveniles or aborted eggs were observed in the control and in the geometric mean measured concentration of 3.43 μg/L during the exposure period of 21 days.

No males or ephippia (winter eggs) were observed in the control or in the geometric mean measured concentration of 3.43 µg/L.

Water quality parameters (i.e. pH-value, dissolved oxygen concentration, total water hardness and temperature) were within the acceptable limits.
Results with reference substance (positive control):
In order to prove the validity of the test system and test conditions at the test facility, an acute immobilisation test according to DIN 38412 L 11 is monthly carried out with potassium dichromate as reference item.
The ECso of the reference item was 1.80 mg/L (95% confidence limits: 1.52 - 2.22 mg/L) after 24 hours and therefore within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to AQS P 9/2 (05/1996) for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.
Validity criteria fulfilled:
yes
Conclusions:
No significant effects on the reproductive output or adult mortality were observed in this study. The No Observed Effect Concentration (NOEC) after 21 days was assessed at 3.43 µg/L, which is the maximum solubility level of the test item in the dilution water and thus equivalent to the nominal loading rate of 100 mg/L.
Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 211. In this study semi-static conditions have been applied.

The measured concentrations of the test item at the start of the exposure intervals were in the range of < LOQM to 5.35 μg/L. At the end of the exposure intervals (48 or 72 h), the measured concentrations of the test item were in the range of < LOQM to 3.60 μg/L. The measured values below 70 % of the LOQM (5.00 μg/L) were taken into account with half of the LOQM (= 2.50 μg/L). The geometric mean measured concentration of the test item was calculated to be 3.43 μg/L and was used for the assessment of the effect levels.

The No Observed Effect Concentration (NOEC) after 21 days was assessed at 3.43 µg/L, which is the maximum solubility level of the test item in the dilution water and thus equivalent to the nominal loading rate of 100 mg/L.

Description of key information

The long-term toxicity to aquatic invertebrates was studied in a GLP compliant OECD 211 assay. The study showed a No Observed Effect Concentration (NOEC) after 21 days of 3.43 µg/L, which is the maximum solubility level of the test item in the dilution water and thus equivalent to the nominal loading rate of 100 mg/L.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
100 mg/L

Additional information