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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:
2002-05-25 to 2002-09-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Solvent control, 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L

- Sampling method: Samples were taken weekly from the newly prepared (solvent) control and from threee test substance concentrations. After 48 hours the same spent media were sampled again.

- Sample storage conditions before analysis: The samples taken were added by syringe through the septum to the sample bottles (glass vials, containing sodium chloride solution, hexane and a stirring bar).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: Stock solutions were prepared at the start of the test and at each medium renewal time. The stock solution vessels were wrapped in aluminium foil until used. Appropriate amounts of test substance were weighed ouy in syringes and dissolved by injection into TBA (Tertiary butyl alcohol). Using a glass syringe, aliquots of 12.5 μL were used to dose the test substance through the septum of the cap of the test vessels.

- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 0.1 mL/L

- Dilution water: DSWL-E water prepared from ground water
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM

- Source: TNO stock culture. Weekly cultures are initiated with 125 daphnids of the same age (circa 1 day) in approximately 4 litres of culture medium. The cultures are maintained at 20+/-1ºC under a 16 h light, 8 h dark regime with a 30 minute transition period. The cultures are discarded after 4 weeks.

- Feeding during test

- Amount: Each 125 mL test vessels received between 40 and 80x10E6 cells/mL of Chlorella sp and 1.5-3.0 mg of yeast. The food was added to the dilution water used to prepare the test media or via a syringe on days between medium renewals.

- Frequency: Daily


ACCLIMATION

- Acclimation conditions: same as test

- Type and amount of food: 4 x 10E9 algal cells (Chlorella sp.) and 0.13 g of yeast in 4 litres

- Feeding frequency: Daily
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
209 mg/L
Test temperature:
19.1-20.7ºC
pH:
7.5-8.0
Dissolved oxygen:
>1.8 mg/L (several low oxygen concentrations were measured in the spent concentrations, however based on the laboratory's opinion this did not affect the results of the study )
Salinity:
Not applicable
Nominal and measured concentrations:
- Nominal test substance concentrations: 0 (control), 0 (solvent control), 0.10, 0.18, 0.32, 0.56, 1.0, and 1.8 mg/L.

The two top doses were above the limit of water solubility to make sure that the test reached water solubility. 

- Over the 3-week test period the measured concentrations averaged 79.4% of nominal. Refer to Table 1 below.

Details on test conditions:
TEST SYSTEM

- Test vessel: Test was carried out in closed silanized glass vessels

- Type (delete if not applicable): closed

- Fill volume: 125 mL

- Aeration: none

- Renewal rate of test solution (frequency/flow rate): 3 times per week (Monday, Wednesday and Friday)

- No. of organisms per vessel: 1

- No. of vessels per concentration (replicates): 10

- No. of vessels per control (replicates): 10

- No. of vessels per vehicle control (replicates): 10

- Biomass loading rate: 1 daphnia per container
 
OTHER TEST CONDITIONS

- Adjustment of pH: no

- Photoperiod: 16 h light-8 h dark


TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: Synthetic medium (DSWL-E)

- Total organic carbon: 1.91 mg/L

- Ca/mg ratio: 2:1

- Culture medium different from test medium: no

- Intervals of water quality measurement: Temperature, pH, and dissolved oxygen were measured every 2-3 days in the spent media.


EFFECT PARAMETERS MEASURED: Mortalities and numbers of young produced were determined at each test medium renewal.


VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY

- Test concentrations: not reported

- Results used to determine the conditions for the definitive study: not reported
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.08 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.26 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: condition
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.25 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.14 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.44 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: condition
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.44 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 0.25-0.37
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
0.45 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
The mean number of offspring in the control and solvent control was 130 and 106, respectively.  The mean number of offspring in the 0.10, 0.18, 0.32, and the 0.56 mg/l treatments was 88, 81, 66, and 22, respectively.  The 0.10 mg/l nominal (0.08 mg/l measured) results were not significantly different from controls so that was determined to be the NOEC for reproduction.  The reproduction LOEC was 0.18 mg/l nominal (0.14 mg/l measured). The 21-day LC50 was estimated to be 0.57 mg/l nominal (0.45 mg/l measured).  All animals died at 1.0 and 1.8 mg/l and 3 died at 0.56 mg/l nominal.   The condition of the organisms was evaluated qualitatively compared to the controls.  Effects on organism condition were observed at 0.56 mg/l, while no effects were observed at 0.32 mg/l or lower.  The NOEC for condition was 0.32 mg/l nominal (0.25 mg/l measured) and the LOEC was 0.56 mg/l nominal (0.44 mg/l measured).

Refer to Table 2 below for test results. Refer to Table 3 for calculation of the time weighted mean (carried out by the present reviewers, not present in original study report).
Reported statistics and error estimates:
The EC50 values and their confidence intervals were calculated using a maximum liklihood fitting procedure on a logistic model.

The LC50 values and their confidence intervals were calculated using a parametric model developed by Kooijman.

The NOEC and LOEC values were determined by comparing treatments with the Controls. Effects on mortality were assessed using a binomial test at the 95 and 99% significance level. Effects on reproduction were assessed using a Dunnett-test at the 95 and 99% significance level.

Table 2. Test results

 Nominal test concentration (mg/L)  Survival at end of test (%)  Cumulative number of live young per female   Cumulative number of live young per female as percentage of Solvent Control
 0 (Control)  90  130  123
 0 (Solvent control)  100  106  -
 0.10  100  88  83
 0.18  100  81*  77
 0.32  100  66*  63
 0.56  70  22*  21
 1.0  0  0*  0
 1.8  0 0*  0

*Significantly different from Solvent Control (p<5%)

Some offspring were found dead in the controls and every concentration at one of the solution replacement times. 
Offspring were monitored for mortality on days 11, 13 and 18.  The mortality was ascribed to the pressure in the closed and completely filled test vessels (droplets noticed on the outside of some of the lid septums).

The mean measured concentration was 79.4% of control, so this number was used in the original study report to calculate measured concentrations.

Validity criteria fulfilled:
yes
Conclusions:
A 21-day EC50 value of 0.30 mg/L has been determined for the effects of the test substance on reproduction of Daphnia magna. A 21-day NOEC of 0.08 mg/L (arithmetic mean), equivalent to 0.085 mg/l (time-weighted mean, derived by reviewer) has been determined for the same endpoint.

Description of key information

A 21-day EC50 value of 0.30 mg/l has been determined for the effects of the test substance on reproduction of Daphnia magna. A 21-day NOEC of 0.08 mg/l (measured arithmetic mean), equivalent to 0.085 mg/l (time-weighted mean, derived by reviewer) has been determined for the same endpoint.

Key value for chemical safety assessment

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

Additional information

A 21-d EC50 value of 0.30 mg/l (measured, arithmetic mean) and NOEC value of 0.080 mg/l (measured, arithmetic mean, equivalent to 0.085 mg/l, time-weighted mean) have been determined for the effects of hexamethyldisiloxane (HMDS, L2, CAS 107-46-0) on reproduction of Daphnia magna. In view of the exposure regime it is likely that the test organisms were exposed primarily to the parent form of the tested substance, although some losses were observed based on measured concentrations in spent media.