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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
14 September 1994 - 19 March 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to GLP requirements and OECD test method. Study is well documented and the data complete.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
Cited as OECD Guide-line 202, part 2 (Daphnia sp., Reproduction Test)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
AHTN

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples taken on day 0, 3 and 21 of the exposure period were worked-up and analyzed by HPLC on the day of sampling. Samples taken on day 19 were stored in the refrigerator at about 4 °C overnight prior to sample work-up and analysis by HPLC. Reserve samples were put in storage at about -20 °C until sample work-up or for a possible future analysis pending a decision from the sponsor.

Test solutions

Vehicle:
yes
Details on test solutions:
Five stock solutions were prepared by diluting an original stock solution of 1250 mg AHTN added to a 100 ml flask brought to volume with dimethylformamide. Test solutions were prepared using 80 µl aliquots from these 5 stock solutions transferred to 1 L flasks with 20 mg Tween 80 then filled to volume with test medium. Two controls were prepared: untreated test medium and untreated test medium with 0.002% Tween 80 and 0.008% dimethylformamide.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
The test was run with the species Daphnia magna bred in the laboratories of RCC under standardized conditions. Stock cultures were maintained in Elendt M7 medium with slight aeration as follows: 100 daphnids with ages of less than 3 days were kept for 7 days. Then, the daphnids with eggs in their brood pouch were transferred into freshly prepared medium and kept for another 7 days. Afterwards, the same animals were transferred into freshly prepared Elendt M7 medium and were kept for an additional 7 days.
The animals were fed daily with Scenedesmus subspicatus equivalent to 0.1 - 0.4 mg C (carbon) per daphnia. Breeding was performed under the same temperature and light conditions as in the test itself. For the test, animals of less than 24 hours of age, originating from about 3-week old adults, were used. The adult animals were separated from the young and put into Elendt M7 medium for about 24 hours prior to the start of the test. The species Daphnia magna was supplied by the University of Sheffield UK in 1992. The quality of the animals used is normally checked twice per year by determination of the EC50 of the reference compound potassium dichromate.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
A range-finding test was performed over a period of six days (test medium renewal on exposure days 1 and 3) with test article concentrations of 1.0, 10, 100 and 1000 µg/l. Solubilisers were 0.008 % DMF and 0.002% Tween 80.

Test conditions

Test temperature:
The room temperature was monitored continuously and ranged from 19.0°C to 21.5°C.
pH:
For each test medium renewal, the pH of the control with and without solubilisers, of the lowest (54.19 µg/l) and highest (804 µg/l) test concentration was measured at the beginning and at the end of the exposure period. The pH varied from 7.7 to 8.9.
Dissolved oxygen:
For each test medium renewal, the oxygen concentration (mg O2/l) of the control with and without solubilisers, of the lowest (54.19 µg/l) and highest (804 µg/l) test concentration was measured at the beginning and at the end of the exposure and ranged from 6.1 to 11.6 mg O2 per litre.
Salinity:
no data
Nominal and measured concentrations:
See table 1 in Remarks on results
Details on test conditions:
Room temperature: 19.0 - 21.5 °C; recorded continuously during the main test
Light: 16 hours/day, about 500-2000 Lux
Test medium: Elendt M7 - Before use, it was aerated until oxygen saturation (8.1 - 9.2 mg/l O2);
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
244 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% CL: 238-249µg/l
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
196 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
401 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
IC50
Effect conc.:
341.3 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks:
parental generation
Remarks on result:
other: 95% CL: 242.7-433.4 µg/l
Details on results:
All test concentration of AHTN and the control with 0.002% Tween 80 and 0.008% DMF were analyzed (as long as the parent animal survived) by HPLC at the beginning and at the end of the first and the last exposure period. Measured test article concentrations ranged from 83.7% to 102.8% of nominal at the start of the first and last exposure period and from 69.6% to 85.1% of nominal at the end of the first and last exposure period (see table 1).

Up to 196.0µg/l no or no significant immobility was observed. At higher concentrations, a concentration related immobility was observed, i.e. 80 % at 400.5µg/l and 100 % at 804.0µg/l (table 2).
The EC50 of the immobility of the parental generation after 21 days of exposure was found to be 341µg/l (95 % confidence limits: 243 - 433µg/l).
Reproduction of young daphnids started between day 8 and day 10 of the exposure period. At the 54.19, 112.9 and 196.0µg/l test concentrations, no significant inhibition was found and the reproductive output ranged from 80% to114 % in comparison to the solvent control group (100%). At 400.5µg/l a statistically significant inhibition of the reproductive output by about 95% was observed. At the 804.0µg/l test concentration no reproduction occurred due to 100% immobility of the parent animals within seven days of exposure.
In conclusion, the NOEC of AHTN on the reproductive capacity in a prolonged daphnia test was found to be 196µg/l, and the LOEC was 401µg/l.
The EC50 of the reproductive output after 21 days was calculated to be 244µg/l (95 % confidence limits: 238 - 249µg/l).
Reported statistics and error estimates:
The reproductive output of the surviving and reproducing animals in the treated solutions was compared to that of the control with 0.002 % Tween 80 and 0.008 % DMF by employing the Dunnett test. The EC50 values of the immobilization rate were estimated by the Logit model inclusive of the 95 % confidence limits.

Any other information on results incl. tables

The end points are based on measured values and table 1 shows the AHTN concentrations in water.

Table 1 AHTN concentrations in water

Nominal
concentration of
test article
(µg/I)
time
(days)
Start
time
(days)
End
% found of nominal Mean % of nominal Mean measured AHTN
concentration (µg/I)
62.5 0   96.4 86.7 54.19
    3 72.4    
  19   102.8    
    21 75.1    
125 0   96.3 90.3 112.9
    3 81.4    
  19   98.4    
    21 85.1    
250 0   89.9 78.4 196
    3 70.3    
  19   83.7    
    21 69.6    
500 0   92 80.1 400.5
    3 70.4    
  19   83.9    
    21 74.1    
1000 0   91.2 80.4 804
    3 69.6    

The immonilisation and the number of surviving/reproducing parents were also determined and are shown in table 2..

Table 2 Survival of parent daphnids exposed to various concentrations of AHTN in a semi-static system for 21 days (values represent number of surviving parent daphnids).

AHTN Concentration
µg/I
% Immobility Number of surviving
and reproducing
parents until day 21
Mean ± s.d.1)
  day 3 day 21    
Solvent control 0 0 8 125 ± 20
Control 0 10 5 125 ± 32
54.19 0 0 10 142±26
112.9 0 10 9 141±15
196 0 0 8 100±33
400.5 0 80 2 4±3
804 0 100 0 0

1) mean reproduction per surviving and reproducing parent

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The total mean offspring in the control >60 per parent animal. The mortality in the control at the end of the test was ≤20 %. The dissolved oxygen concentration remained >3 mg/l.
Conclusions:
The test meets the criterion for the validity of the test. NOEC (day 21): 196 µg/l. LOEC (day 21): 401 µg/l.
The EC50 of the reproductive output after 21 days of exposure was calculated to be 244 µg/1 with the 95 % confidence limits of 238 - 249 µg/l.
The EC50 of the immobility of the parental generation after 21 days of exposure was calculated to be 341µg/l with the 95 % confidence limits of 243 - 433µg/l.
Executive summary:

AHTN was tested at nominal concentrations ranging from 62.5-1000 µg/l in Daphnia magna for reproduction and survival over 21 days in a semi-static test following OECD guideline 202, part II. Mean measured concentrations in 0.002% Tween 80 and 0.008% dimethylformamide were determined to be 54.19, 112.9, 196.0, 400.5, and 804.0 µg/l using HPLC. Test media were renewed several times during the exposure period. No significant immobility was reported at concentrations up to 196 µg/l but 80% immobility was noted at 400.5 µg/l and 100% was reported at 804 µg/l. Reproductive output was not significantly affected at 54.19-196.0 µg/l (ranging from 80-114% compared to solvent control) but was significantly inhibited (p<=0.05) at 400.5 µg/l by about 95%. No reproduction occurred at the highest concentration. The 21-day NOEC and LOEC values of the reproductive output (expressed as mean measured concentrations) were 196 and 401µg/l, respectively. The 21-day EC50 values (with 95% confidence limits) for reproductive output and immobility of the parental generation were 244 µg/l (238-249 ug/l) and 341µ/l (243-433 ug/l), respectively.