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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:
26 July to 18 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Dilution water control, solvent control (dimethylformamide 0.1 mg/L) and a nominal concentrations of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L of the test substance
- Sampling method: Analytical samples taken at 0, 2, 9, 12, 19, 21 days and analysed by HPLC-UV instrumentation
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A nominal 1000 g/L stock solution of the test substance was prepared by the addition of 5.0 g (nominal) of the test substance to a 5 ml volumetric flask which was made up to the mark with dimethylformamide. The solvent concentration of this stock solution was approximately 10% v/v. The top concentration of 100 mg/L was prepared by the addition of 200 µL of the stock solution to 2000 mL of dilution water. This stock solution was stirred vigorously at approximately 30°C to aid the solubility of the test substance. The solution was then cooled to 20°C after which it was filtered through a 1.2 µm GFC filter paper and on all occasions observed to be a clear and colourless solution. The highest test concentration used this solution without any further alteration.

The remaining test solutions were prepared from the 100 mg/L solution, by the addition of appropriate volumes to volumetric flasks which were subsequently made up the mark with dilution water and stirred using a magnetic stirrer. A solvent control was prepared by the addition of 10 µl/L of DMF to dilution water. Equalising additions of DMF were made where applicable to give a final DMF concentration of 10 µl/L in each test concentration. The control consisted of dilution water only. The resultant solutions were clear and colourless.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia Magna
- Age at study initiation (mean and range, SD): < 24 hrs old obtained from a single culture vessel.

- Source: obtained from continuous laboratory cultures held at Brixham Environmental Laboratory
- Age of parental stock (mean and range, SD): Parent animals were 14 days old and had been maintained with a twice weekly renewal of reconstituted water medium since birth.

ACCLIMATION
- Acclimation conditions (same as test or not): yes
- Type and amount of food: fed on a mixed algae diet of Chlorella vulgaris, CCAP 211/12 and Pseudokirchneriella subcapitata, strain CCAP 278/4. The algal cultures were harvested, centrifuged and measured for cell density using a Coulter Counter. The algal cells were then re-suspended in D. magna culture medium (reconstituted water), stored in a refrigerator, in the dark, and used within two weeks. The algal stocks, once re-suspended, were analysed for total organic carbon content. The feeding was designed so that each vessel receive a nominal 0.06 mg C/day of each algal suspension.
- Feeding frequency: fed daily ad libitum
- Health during acclimation (any mortality observed): no mortality observed

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS: diploid parthenogenesis
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
total hardness of the control, solvent control and highest exposure concentration were measured on 3 occasions during the test.
Test temperature:
19.4 to 20.7 °C
Temperature values were determined daily in an additional replicate vessel (without D. magna) using calibrated thermometer. Hourly temperature measurements were also recorded automatically in the additional vessel using an electronic system.
pH:
pH of 'on' solutions: 7.7 to 8.1
pH of 'off' solutions: 7.5 to 8.2
Dissolved oxygen:
DO of 'on' solutions: 6.4 to 9.8 mg/L
DO of 'off' solutions: 6.6 to 9.8 mg/L

Range was from 6.4 to 9.8 mg/L equivalent to a range of 70 to 108% of the air saturation (9.1 mg/L = 100%) for freshwater at a nominal temperature of 20 C
Nominal and measured concentrations:
Nominal concentrations: Dilution water control, solvent control, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers
- Type (delete if not applicable): covered with loose fitting glass lids
- Material, size, headspace, fill volume: 80 ml of test solution providing a depth of approx. 50 mm
- No. of organisms per vessel: 1 impartially selected (P0 generation)
- No. of vessels per concentration (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt's M4 D. magna medium.
- pH: 7.8
- Ammoniacal Nitrogen as N: < 0.002 mg/L
- Solid Susptended at 105°C: < 3.00 mg/L
- Dissolved Organic Carbon: 0.21 mg/L
- COD: < 12 mg/L
- Organochlorine pesticides: < 0.1 µg/L
- Organophosphorus pesticides: < 0.01 µg/L
- PCBs: < 0.001 µg/L
- Alkalinity: 27 mg CaCO3/L
- Hardness: 221 mg/L as CaCO3
- Culture medium different from test medium: no
- Intervals of water quality measurement:

Final nominal concentrations in the media:
- NaHCO3: 64,8 mg/L
- CaCl2. 2H2O: 293.8 mg/L
- MgSO4.7H2O: 123.3 mg/L
- KCl: 5.8 mg/L
- Na2SiO3.5H2O: 0.747 mg/L
- NaNO3: 0.02274 mg/L
- KH2PO4: 0.0143 mg/L
- K2HPO4: 0.0184 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: no, measurements were undertaken for pH at the start and end of the test and on each renewal occasion. On each occasion the pH and DO concentration of the control, solvent control and highest exposure concentration were measured. For the 'on' solutions, the excess solution remaining after filling the vessels was used. After renewal, one 'off' replicate of the control, solvent control and highest exposure concentration were measured. These pH and DO measurements were taken after transfer of the P0 D. magna and countng of offspring.
- Photoperiod: 16 hours light: 8 hours dark with 20 minute dawn and dusk transition periods
- Light intensity: 11.6 µ Einsteins m2/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations were made daily from exposure Day 0 for the presence of live and dead offspring (termed F1 generation) in each vessel. The test solutions were prepared on Day 0 and were renewed every Monday, Wednesday and Friday throughout the study. In each renewal day, new solutions were dispensed to a second set of test vessels and the surviving P0 generation were transferred to the new solutions. Both old ('off') and new ('on') solutions were sampled, as appropriate, for physical and chemical analysis and the F1 generation were removed from each vessel and counted. The number of live and dead F1 offspring were recorded. At the end of the test, (apex of helmet to base of spine) of each survivng P0 Daphnia was measured, using a microscope with an eyepiece graticule previously calibrated with a stage micrometer.

Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
26.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% CL
Remarks:
25-28 mg/L
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
69.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL
Details on results:
- although all freshly prepared 'on' solutions were within 100± 20% of the nominal values, on the last renewal occasion, exposure day 19, the solutions were dispensed into the test vessels incorrectly. On day 19, the nominal 0.32 mg/L received the solution prepared at 32 mg/L, the nominal 1.0 mg/L received the solution prepared at 10 mg/L, nominal 10 mg/L received the solution prepared at 1 mg/L and the nominal 32 mg/L received the solution prepared at 0.32 mg/L. The control, solvent control, 3.2 and 100 mg/L were dispensed to the correct test vessels.
This error only affected the final 48 hrs of the study, a clear dose response was observed prior to this point, all other analysis points showed measured concentration of the test substance to be 100 ± 20% of the nominal values (with the exception of the nominal 1.0 mg/L on day 12 off solution which was 65% of nominal concentration) and statistical analysis shows that using the full 21 days reproduction data (rather than data up until Day 19) provides a more precautionary result. Therefore nominal concentrations were used for the calculation and reporting of results. Due to the LOQ (0.34 mg/L) it was not possible to quantify the concentration of the substance in the low test concentrations so the nominal concentration was use for the calculation and reporting of results.

The measured concentrations in the 'on' test solutions ranged from 84% to 110% of the nominal value, with the corresponding 'off' values, excluding day 21, ranged from 65 to 93% of nominal. The measured concentrations in the 'off' test solutions for day 21 ranged from 1.2 to % of nominal. The analysis of the triplicate samples, taken from the nominal 1.0 and 10 mg/L solutions showed very good consistency.

- Mortality of control:
* there were 2 mortalities in the solvent control on exposure day 21, prior to the mortalities both animals were observed to be very pale and bloated. On days 18 to 20 one replicate of the 10 mg/L concentration appeared pale in comparison to other replicates at the same concentration; this animal was recorded as a mortality (10%) on Day 21. Aborted eggs were observed in all replicates of the 32 mg/L concentration of Day 21. From exposure Day 5 to 21 animals in the 100 mg/L concentration were observed to be pale and small in comparison to those in the control. On Day 18, one replicate of the 100 mg/L concentration was observed to have aborted eggs. A total of 8 mortalities (80%) were observed in the 100 mg/L which occurred on exposure Days 10, 13, 15,16, 19 and 20.
* there were no mortalities in the control and 2 mortalities (20%) in the solvent control after 21 days; this satisfies the guideline validity criteria of ≤20% mortality in the controls at the end of the test.


Reported statistics and error estimates:
Reproduction and length data for each individual P0 generation daphnia were entered into electronic data files and statistically analysed using Minitab. The control and solvent control were assessed for normality (Anderson-Darling) and found to be normal and so were assessed for quality of variances using the F-test, variances were found to be equal. Consequently statistical differences between the control and the solvent control were assessed for significance using a 2 sample T-test. No statistical significant difference (p< 0.05) was found between the control and solvent control. All subsequent comparison was made against the solvent control.
Reproduction:
The data were assessed for normality (Anderson-Darling) and were found to be non-normally distributed, Therefore, the data were assessed for equality of variances using Levene's test. The treatment variances were found to be equal and so significant differences from the solvent control were analysed using a One-way Analysis of Variance and Dunnett's test.
EC50 values were calculated using Linear Interpolation method using the US EPA program ICPIN.

Length:
The data were assessed for normality (Anderson-Darling) and were found to be non-normally distributed, Therefore, the data were assessed for equality of variances using Levene's test. The treatment variances were found to be equal and so significant differences from the solvent control were analysed using a One-way Analysis of Variance and Dunnett's test.

Reproduction data

 Nominal conc. of Daltolac R 159         Total number of offspring per P0 D. magna                    

 Mean per P0

D. magna

 SD  Total offspring  Mean offspring as % of solvent control
 (mg/L)  1  3  4  5  6  7  8  9  10        
 control  145 151 141  132  141  146  144  141  148  143   143  5.1  1432  -
 solvent control  136  151  151  M 145   135  156  150  144  146  7.5  1168  -
 0.32  147  146  144  137  135  142  132  138  140  134  140  5.2  1395  96
 1.0 134  143  134  138  134  134  148  133  147  129   137*  6.4  1374  94
 3.2  125  129  127  122  137  138  126  124  115  137  128* 7.4   1280  88
 10  133  142  129  132  134  M  125  128  129  121  130*  6.0  1173  89
 32  59  64  64  54  49  53  45  50  48  67  55*  7.7  553  38
 100  M  M  M 0 0  0*  0  0  0

* statistically significant difference between treatment and solvent control (p< 0.05)

Adult length measurements

 Nominal conc. of Daltolac R 159         Length in graticule scale divisions (day 21) per P0 D. magna        Mean length  SD   Mean $
 (mg/L)  1  3  4  5  6  7  8  9  10         
 control 57 55 57  56  54  54  57  54  56  53  55  1.5   4.3
 solvent control 56 57 56 56 M 55  54  56  57  56  1.0    4.3
 0.32 56 55 56 56 54 57 57 57 57 56 56  1.0    4.3
 1.0 55 57 56 56 55 57 53 56 54 54 55  1.3    4.3
 3.2 54  56 57 56 56 57 55 55 54 57 56 1.2  4.3
 10 58  56 56 57 56 M 57 56 55 55  56 1.0  4.3
 32 55  56 55 56 53 56  55 56 55  56  55 0.9  4.3
 100 M  M M M M M M M 45 43  44*  1.4   3.4

$ length in mm (1 mm = 12 scale divisions)

* statistically significant difference between treatment and solvent control (p< 0.05)

Validity criteria fulfilled:
yes
Conclusions:
Chronic toxicity to invertebrates has been investigated in a reproduction test with Daphnia magna following OECD guideline 211 using semi-static exposure. The 21d-NOEC was reported as 0.32 mg/l (nominal) for reproduction. Exposure concentrations were verified by mean of HPLC-UV/VIS.

Description of key information

The 21 day-NOEC in a OECD 211 guideline study is 0.32 mg/l (nominal) for reproduction.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.32 mg/L

Additional information

Chronic toxicity to invertebrates has been investigated in a reproduction test with Daphnia magna following OECD guideline 211 using semi-static exposure. The 21d-NOEC was reported as 0.32 mg/l (nominal) for reproduction. Exposure concentrations were verified by mean of HPLC-UV/VIS.