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Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 April 2007-24 April 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to GLP requirements and OECD draft guideline. Study is well documented and the data complete.
Qualifier:
according to
Guideline:
other: OECD Draft Guideline for life cycle test with Acartia tonsa (2004)
Deviations:
yes
Remarks:
Only the last part of the test described in the draft Guideline was performed, i.e. exposure from eggs to copepodites (F0 generation) - totally 6 days. Specific adaptations were made to prevent volatilisation and accumulation of organic debris.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Samples from parallel flasks were harvested at the start of the test, every 24 hours during the test and at the end of the test.
Vehicle:
yes
Details on test solutions:
Test concentrations were 5.5, 11, 22, 44, 88, 176 and 352 µg/l, with four replicates per concentration and 6 replicates in the control as well as in the solvent control.
Test organisms (species):
other aquatic crustacea: Acartia tonsa
Details on test organisms:
A strain of the marine copepod Acartia tonsa (DANA), Copepoda, Crustacea collected in the North Sea has been cultured at DHI since 1987 according to the DHI Standard Operation Procedure.
The test was initiated with eggs taken from this culture, and the larval development rate was calculated from the number of surviving larvae (copepodites compared to the sum of nauplii and copepodites).
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
6 d
Post exposure observation period:
A preliminary range-finding test was performed at 2.5 μg/L and 25 μg/L. It showed that there was no significant effect on growth (length) at 25 μg/L. Therefore, the following test concentrations were chosen for the final test: 5.5; 11; 22; 44; 88; 176 and 352 μg/l.
Test temperature:
The test was carried out at 20 ± 1.0°C in a climate room with a daily light/dark period of 16:8 hours.
Salinity:
Salinity: 32.5‰ (natural seawater)
Nominal and measured concentrations:
The actual test concentrations were >80% of nominal during the whole study, implying that the nominal concentrations can be used for the calculations.
Details on test conditions:
For the test 110-ml glass flasks were used, closed with screw caps with Teflon seals. The algal concentration was readjusted daily to 7,000 cells/ml and the flasks were slowly rotated to keep the algae from settling. This was confirmed to be the optimal design to prevent the formation of organic debris and the loss of test substance by sorption and volatilisation.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
6 d
Dose descriptor:
NOEC
Effect conc.:
22 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Larval Development Rate (LDR)
Remarks on result:
other: This effect parameter was the most sensitive of all studied endpoints (Recovery of added eggs, Hatching, Nauplii size (length), Copepoddites size (length) and LDR)
Duration:
6 d
Dose descriptor:
LOEC
Effect conc.:
44 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Larval Development Rate (LDR)
Duration:
6 d
Dose descriptor:
EC10
Effect conc.:
28 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Larval Development Rate (LDR)
Remarks on result:
other: 95% CL: 17-36.8µg/L
Duration:
6 d
Dose descriptor:
EC50
Effect conc.:
72 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Larval Development Rate (LDR)
Remarks on result:
other: 95% CL: 60.5-87.2µg/L
Duration:
6 d
Dose descriptor:
other: EC90
Effect conc.:
182 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Larval Development Rate (LDR)
Remarks on result:
other: 95% CL: 135-330µg/L

The larval mortality in the controls was below the quality criterion of 30 %: 19.5 and 15.3 % in the control and solvent control, respectively. The larval development ratio (LDR) was 69% in the control and 75% in the solvent control. The NOEC(LDR) was 22μg/l with an inhibition of 3% as compared to the solvent control, whereas the inhibition was 20% in 44 μg/l (LOEC). At 176 μg/l no copepodites were observed and the length of the remaining nauplii was significantly smaller than in the controls. Moreover no copepodites were observed. Up to and including 88 μg/l, there was no impact on growth. The EC10(LDR) was 28.2 μg/l (95% confidence interval 17.0 – 36.8 μg/l) and the EC50 was 71.7 μg/l (95% confidence interval 60.5 – 87.2 μg/l). The results are suumarised in table 1.

Table 1 Test results from larval development tests

Parameter NOEC (μg/L) LOEC (μg/L) EC10 (μg/L) EC50 (μg/L)
Recovery of added eggs 88 176
Hatching 88 176
Nauplii size (length) 88 176
Copepoddites size (length) 88 176
Larval Development Rate (LDR) 22 44 28 72
Validity criteria fulfilled:
yes
Conclusions:
The 5d-EC10 (larval development ratio) was 28 μg/l and the 6d-NOEC 22 μg/l. The 6d-EC50 was 71.7 μg/l (60.5 – 87.2 μg/l).
Executive summary:

The chronic toxicity to marine copepod Acartia tonsa was tested according to the OECD Draft Guideline for Testing of Chemicals (2004) describing a life cycle test, with specific adaptations to prevent volatilisation and accumulation of organic debris (Bjørnestad 2007). For this test AHTN was mixed with radio-labelled [phenyl - 14C(U)]-AHTN (1%) with a radiochemical purity of 94.8%. Ethanol was used as a solvent. Test concentrations were 5.5, 11, 22, 44, 88, 176 and 352 µg/l, with four replicates per concentration and 6 replicates in the control as well as in the solvent control. For the test 110-ml glass flask were used, closed with screw caps with Teflon seals and filled almost completely with natural seawater, salinity 32.5‰ and test substance leaving a headspace of 5 ml. The test was carried out at 20 ± 1.0°C in a climate room with a daily light/dark period of 16:8 hours. One day before addition of the eggs, the flasks were saturated with the appropriate test concentration. At the initiation of the test, fresh solutions were added to the bottles. The test was initiated with approximately 80 eggs in each flask and approx. 7,000 cells/ml of the algae Rhodomonas salina. The algal concentration was readjusted daily to 7,000 cells/ml and the flasks were slowly rotated to keep the algae from settling. This was confirmed to be the optimal design to prevent the formation of organic debris and the loss of test substance by sorption and volatilisation. Samples of the test solution for LSC were taken daily. After 5.5 days the number of nauplii, copepodites and non-hatched eggs was counted and the lengths of the larvae was measured.

The actual test concentrations were >80% of nominal during the whole study, implying that the nominal concentrations can be used for the calculations. The larval mortality in the controls was below the quality criterion of 30%: 19.5 and 15.3 % in the control and solvent control, respectively. The larval development ratio (LDR) was 69% in the control and 75% in the solvent control. The 6d-NOEC(LDR) was 22 μg/l with an inhibition of 3% as compared to the solvent control, whereas the inhibition was 20% in 44 μg/l (LOEC). At 176 μg/l no copepodites were observed and the length of the remaining nauplii was significantly smaller than in the controls. Moreover no copepodites were observed. Up to and including 88 μg/l, there was no impact on growth. The 6d-EC10(LDR) was 28.2 μg/l (95% confidence interval 17.0 – 36.8 μg/l) and the 6d-EC50 was 71.7 μg/l (95% confidence interval 60.5 – 87.2 μg/l).

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.
Reason / purpose:
reference to same study
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
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.
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 (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.
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 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
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.

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

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.

Description of key information

The NOEC of the freshwater invertebrates was obtained from the reproduction study with Daphnia magna and the NOEC for the marine water invertebrates was obtained from the larval development study on the marine copepod Acartia tonsa. Both studies were performed according to international guidelines and in compliance with GLP. Concentrations were measured to ensure the stability of exposure. The 21d-NOEC (reproduction) for D. magna was 0.196 mg/L;

The 6d-EC10(larval development) for A. tonsa was 0.028 mg/l; NOEC was 0.022 mg/L. As a worst-case approach, the NOEC (lowest value) of the A. tonsa was selected.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
196 µg/L
EC10, LC10 or NOEC for marine water invertebrates:
22 µg/L

Additional information

A. tonsa proved to be a very sensitive species upon AHTN exposure. This study was initiated as a result of the publication of Wollenberger et al (2003) of earlier results with A. tonsa, resulting in a very low 5d-NOEC for larval development. As this test showed a number of methodological deficiencies. The new test was (Bjornestad 2007) was assessed as a Reliability 1 study and the earlier results of Wollenberger were disregarded.

It must be noted that the EU Risk Assessment Report used the 6d-EC10 value of 28 µg/L for the larval development rate instead of the NOEC. As a worst-case approach for this CSA, the NOEC (lowest value) of the A. tonsa has been selected.