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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:
01/09/2000 - 16/01/2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 211 (OPPTS 850.1300) in compliance with GLP, without deviations that influence the quality of the results.
Qualifier:
according to
Guideline:
EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: nominal: 0.02, 0.05, 0.2, 0.4 and 0.8 mg/l
- Sampling method: During the prolonged toxicity study, the following sampling regime was applied for confirmation of test concentrations based on measurement of radioactivity:

Number Singular samples from each vessel, i.e. two samples per concentration.
Frequency On days 0, 8, 14 and 21 from all test concentrations.
Volume 2 ml, corresponding with a radio activity of ca. 500 dpm.
Storage The samples were measured on the day of sampling.
Additional sampling was performed on the dosing before and after addition of algal cells for feeding on days 7 and 8, respectively.

During the prolonged toxicity study, the following sampling regime was applied for confirmation of the stability of the test substance in the test solutions based on Thin Layer Chromatography (TLC):

Frequency: On days 0, 8, 14 and 21 samples were taken from each vessel, i.e. two samples per concentration. The samples were stored in a deep-freeze until analysis. Per concentration the samples were pooled before extraction.
Volume: ca. 500 ml.
- Sample storage conditions: No data
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The standard test procedures required generation of test solutions which should contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturb the test system should be prevented (e.g. film of the test substance on the water surface).

The water solubility of OTNE is 2.68 mg/L. The density is 0.963 g/ml. Stock solutions were prepared in acetone. Daily, 0.5 ml of these stocks were added to 5 liters of test medium providing the required test concentrations. Each concentration consisted of a mixture of radio labelled OTNE14C and unlabelled OTNE. The concentration of radio-labelled OTNE-14C corresponded with activities ranging between 250 and 280 dpm per ml.

- Controls: yes, blanks and vehicle controls
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 50 ul/l
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus, 1820
- Source: No data
- Age of parental stock (mean and range, SD): For the test selection of young daphnids with an age of < 24 hors form a batch of daphnids of at least 10 days old.
- Feeding during test: The dosing rate of algal suspension was 10 ml per test vessel per day. This corresponded with 20x10^8 cells. Per daphnia the feeding thus approximated 0.5 x10^8 cells per day. According to the intern NOTOX project 320783 a daily ration of 0.5 x 10^8 cells per daphnia corresponds with 0.15 or 0.16 mg C/Daphnia/day.

ACCLIMATION
- Acclimation period: No data
- Acclimation conditions (same as test or not): No data
- Type and amount of food: No data
- Feeding frequency: No data
- Health during acclimation (any mortality observed): Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and without delay in the production of the first brood.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: No data
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
180 mg/l CaCO3
Test temperature:
20.5 - 21.8 degrees Celsius.
pH:
Blank control: 7.8 - 8.1; solvent control: 7.6 - 8.1; OTNE treatments: 8.0 - 7.2.
Dissolved oxygen:
8.6 - 9.4 mg/l
Nominal and measured concentrations:
Nominal: 0.02, 0.05, 0.2, 0.4 and 0.8 mg/l
Measured (0d): 0.020, 0.055, 0.193, 0.400 and 0.820 mg/l
Measured (7d): 0.012, 0.037, 0.135, 0.276 and 0.589 mg/l
Measured (14d): 0.010, 0.022, 0.083, 0.281 and 0.322 mg/l
Measured (21d): 0.016, 0.035, 0.120, 0.247 and 0.477 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: stainless steel, 1.5 litre per concentration
- Aeration: No data
- Renewal rate of test solution (frequency/flow rate): flow rate: 200 ml/hour, residence time 7.5 hour
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Major nutrients: freshly prepared ultra-pure water (tap water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges: Milli-Q water; Millipore Corp., Bedford, Mass., USA).
- Contains (mg/L):

CaC12.2H20 203
MgSO4.7H20 86
NaHCO3 45
KCI 4.0

Medium M7: trace elements, macro nutrients and vitamins are added to freshly prepared ISO-medium to reach the following
concentrations:
Trace elements.(mg/L):
B 0.125
Fe 0.05
Mn 0.025
Li, Rb and Sr 0.0125
Mo 0.0063
Br 0.0025
Cu 0.0016
Zn 0.0063
Co and I 0.0025
Se 0.0010
V 0.0003
Na2EDTA.2H2o 2.5

Macro nutrients (mg/L):
Na2Si03.9H20 10
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18

Vitamins (Ng/1):
Thiamine 75.0
B12 1.0
Biotin 0.75

- Total organic carbon: < 2 ppm
- Culture medium different from test medium: No
- Intervals of water quality measurement: three times a week


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16/8 hours light/dark
- Light intensity: no data


EFFECT PARAMETERS MEASURED (with observation intervals if applicable):

Condition: Every workday (except weekends). Dead daphnids are removed when observed.
Presence of eggs in the brood pouch: Every workday (except weekends).
Body length: At the end of the test.
Appearance of first brood: When observed.
Newborn daphnids: Every workday (except weekends), the numbers of living newborn young are counted and recorded. Thereafter the newborn young are removed.
Presence of unhatched eggs: When observed.
Incidence of immobility: When observed.


VEHICLE CONTROL PERFORMED: yes
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 0.448 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: radioactive compound
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.448 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: radioactive compound
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.285 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: radioactive compound
Basis for effect:
reproduction
Remarks on result:
other: 95% CL: 0.122 - 0.663 mg/l
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.096 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: radioactive compound
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.028 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: radioactive compound
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.244 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: radioactive compound
Basis for effect:
other: body length
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.096 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: radioactive compound
Basis for effect:
other: body length
Details on results:
- Mortality of parent animals: 0 - 7.5%
- No. of offspring produced per day per female: 1.5 - 3.3
- Body length and weight of parent animals: 2.92 - 3.91 mm
- Time to first brood release or time to hatch: 8 days
- Brood size: 31.5 - 68.7 young
- Other biological observations: Body length significantly lower in OTNE treatments than in controls.
- Effect concentrations exceeding solubility of substance in test medium: No
Reported statistics and error estimates:
Determination of L(E)C„ values and respective parameters:
When applicable the L(E)C50 (mortality and or immobilisation) was determined using maximum likelihood estimation method with the probits of the relative number of affected daphnids as function of the logarithms of the corresponding concentrations (SAS v. 6.12). The cumulative reproduction values expressed as percentages of the mean cumulative reproduction of the solvent control group after 21 days was set out against the logarithms of the corresponding concentrations of the test substance. With a test concentration related effect, calculation of the EC50-value was based on the respective curve using regression analysis. If possible, also the EC10 was determined

Statistical analysis:
The cumulative numbers of living young per parent were analysed statistically as a measure of reproductive capacity. The cumulative numbers of young recorded at the various test concentrations and the different time points during the test period were statistically tested using an ANOVA test followed by a mean comparison test (Dunnett's test and Tukey HSD test, SAS, v. 6.12). The overall LOEC and the overall NOEC were determined on basis of these statistics. In addition, the MATC was calculated as the geometric mean of the LOEC and the NOEC.

Measured concentrations of OTNE in water samples

The OTNE concentrations measured in the samples taken on day 0 were in agreement with thetarget concentrations. However, measurement of OTNE-14C levels in samples taken on day 2 showed recoveries of ca. 50 %. On day 7 samples were taken after the dosing left the mixing vessels and before it entered the test vessels. Also these samples contained about 50% of the target concentrations (see Appendix I). On day 8 samples were taken from the 24-hour old stocks. These measurements confirmed that the original concentrations were in agreement with the targets (see Appendix I). The algal cells for feeding were added to the mixing vessels before the dosing entered the test vessels. These results indicate that the presence algae decreased the actual concentrations of OTNE by ca. 50%, probably due adsorption.

The recoveries of OTNE in the samples taken during the period from days 2 to 21 indicated that, the concentrations remained rather stable within the ±20% window.

Determination of the stability of the radio-chemical purity of OTNE-14C by TLC

The water samples were extracted in three successive steps with dichloromethane (DCM). The TLC chromatograms show tailing in all samples leaving 50 to 70% of radiochemical activity corresponding with the retention time of the original molecule. However, the same tailing was observed in the TLC chromatogram of a sample taken from the original batch of OTNE- 4C (Appendix VII, lane #1). In fact, the pictures resemble closely the chromatogram of OTNE-14C before purification. Hence, although possible degradation products were probably present in the samples taken from the test solutions, these corresponded with those normally found in the original batch after a certain period of storage. Hence, these results indicate that LSC measurement of OTNE-14C concentrations is a reliable method for the determination of actual OTNE concentrations.

Parental mortality:

Less than 10 % mortality was recorded in the controls. The mortality ranged between 2.5 and 7.5 % in the OTNE treated test solutions. Hence, the mortality rates recorded were not significant at any of the OTNE concentrations tested.

Reproduction:

The reproductive capacity of the three higher OTNE treatments was affected. Traditional statistical analysis (SAS, v. 6.12) showed significant inhibition of reproductive capacity at OTNE concentrations of 0.096 mg/L and higher, according to the Dunnet-t test and the Tukey method of multiple comparison (P=0.05). During the total exposure period of 21 days, no significant (>10%) immobilisation (including mortality) of newborn young was observed at any of the test concentrations. The first broods were observed on or before day 9 at all treatments. No biologically significant numbers of unhatched eggs were observed during the reproduction phase in any of the test groups.

Body length:

Statistical analysis showed that the body lengths were significantly lower than the mean body length of the solvent-control at OTNE concentrations of 0.244 and 0.448 mg/L according to the ANOVA procedure with the Tukey method of multiple comparison and the Dunnet-t test (P=0.05).

Validity criteria fulfilled:
yes
Remarks:
Parent mortality <20%, offspring survival > 60%
Conclusions:
The 21d-NOEC for reproduction of OTNE to Daphnia magna is 0.028 mg/l, the 21d-EC50 for reproduction is 0.285 mg/l.
Executive summary:

The toxicity of OTNE to Daphnia magna was investigated in a 21-day chronic reproduction test according to EPA OPPTS 850.1300 / OECD Guideline 211 under GLP. Daphnia were exposed to 14C-labeled OTNE in a flow-through test at nominal concentrations of 0.02, 0.05, 0.2, 0.4 and 0.8 mg/l. Validity criteria for the test were met. The 21d-EC50 for parental survival was found to be > 0.448 mg/l, the 21d-EC50 for reproduction was found to be 0.285 (0.122 -0.663) mg/l, the 21d-NOEC for reproduction was found to be 0.028 mg/l (lowest NOEC observed) based on mean measured concentrations.

Description of key information

 The toxicity of OTNE to Daphnia magna was investigated in a 21-day chronic reproduction test according to EPA OPPTS 850.1300 / OECD Guideline 211 under GLP. Daphnia were exposed to 14C-labeled OTNE in a flow-through test at nominal concentrations of 0.02, 0.05, 0.2, 0.4 and 0.8 mg/l. Validity criteria for the test were met. The 21d-EC50 for parental survival was found to be > 0.448 mg/l, the 21d-EC50 for reproduction was found to be 0.285 (0.122 -0.663) mg/l, the 21d-NOEC for reproduction was found to be 0.028 mg/l (lowest NOEC observed) based on mean measured concentrations.

Key value for chemical safety assessment

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

Additional information