Pentasodium 7-(4-(4-(5-amino-4-sulfonato-2-(4-((2-(sulfonato-ethoxy)sulfonyl)phenylazo)phenylamino)-6-chloro-1,3,5-triazin-2-yl)amino-2-ureidophenylazo)naphtalene-1,3,6-trisulfonate

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Aug 1997 to 28 Jan 1998

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 (incl. QA statement)

Specific details on test material used for the study:

Identity: FAT 40'549/A
Batch No: TV1
Expiration date: 01 Jan 2001
Stability in water: for atleast 48 hours
Solubility in water: approximately 90 mg/L at 20 degree celcius.
Aggregate state under storage conditions: solid (powder)
Colour: yellow brown.
Storage conditions: at room temperature
Safety precautions: Routine hygenic procudures are sufficient to assure personnel health and safety.

Analytical monitoring:

yes

Details on sampling:

The pH-values and dissolved oxygen concentrations in the test media were measured in all test concentrations and in the control at the beginning and end of each treatment period, but only the pH-values and oxygen concentrations of the lowest and highest test concentration and the control are reported since in all test media pH-values and oxygen concentrations were in the same range. At the same times the water temperature was measured in one control beaker.

The mortality of adults and the number of young were recorded at least three times per week before renewal of test media. Dead animals and offspring were removed at the renewal of the test media.

Vehicle:

no

Details on test solutions:

At each treatment a stock solution of nominal 100 mg/l was prepared just before each test medium renewal by dissolving 25, respectively 35 mg of the test substance into 250, respectively 350 ml test water. Then adequate volumes of the intensively stirred stock solution were mixed into test water to obtain the desired test concentrations before introduction of the test animals.

The nominal concentrations tested were 0.20, 0.63, 2.0, 6.3 and 20 mg test substance/L. Additionally, a control was tested in parallel (test water without addition of the test substance).

The test concentrations were based on the results of a range finding test and on the results of a first experiment.

Test organisms (species):

Daphnia magna

Details on test organisms:

The study was performed with females of a clone of the species Daphnia magna Straus. The clone was originally supplied by the University of Sheffield/UK in 1992, defined from the supplier as clone 5. Since this date the clone is bred in the laboratories of RCC under similar temperature and light conditions as in the test, and in the same kind of test water as used in the test. At the start of the test the young daphnids used were less than 24 hours old.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

2.5 mmol/L (= 250 mg/L) as CaCO3

Test temperature:

20 °C

pH:

7.6 - 8.1

Dissolved oxygen:

8.4 - 9.1 mg/L

Salinity:

Alkalinity: 0.9 mmol/L

Nominal and measured concentrations:

The nominal test concentrations were 0.20, 0.63, 2.0, 6.3 and 20 mg test substance/L and a control.

In the test media the test substance degraded into a defined reaction product. The mean measured concentrations in the samples from the freshly prepared test media ranged from 76 % to 90 % of the nominal concentrations, if the quantification is based on the main compound. If the quantification is based on the sum of main compound and degradation product, the mean recoveries in the non-aged samples were nearly identical. However, in the 48 hours-aged samples the mean measured concentrations had decreased to 17 - 35 % of the nominal values, in the 72 hours-aged samples to 4 - 32 % of nominal (quantification based only on the main compound). The decrease of the concentrations during the test medium renewal periods of 48 and 72 hours was obviously caused by degradation of the main compound due to hydrolysis.

If the quantification of the test substance in the aged samples is based on the sum of main compound and degradation product, 67 - 76 % of nominal were found in the samples from the test media without food particles.

Details on test conditions:

The study was started (Day 0) with 10 daphnids per test concentration and control. Each test animal was kept individually in a 100 ml glass beaker containing 80 ml test medium. The beakers were covered with watch-glasses. The test was performed in a temperature controlled room.
Light conditions: a 16-hour light to 8-hour darkness photoperiod. Light intensity at light period: approximately 300 - 800 Lux.
Test water: The test was conducted in reconstituted water with an initial pH of 7.9 ±0.3. Before use, the test water was aerated until oxygen saturation. During the test the test media have not been aerated.

Reference substance (positive control):

not specified

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: main compound and degradation product

Basis for effect:

other: reproduction rate and survival rate

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1.55 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: main compound and degradation product

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

4.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: main compound and degradation product

Basis for effect:

other: survival rate

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: main compound and degradation product

Basis for effect:

reproduction

Remarks on result:

other: 0.03-2.1 mg/L

Details on results:

Survival of adults:
In the control and at the test concentrations up to and including nominal 2.0 mg test substance/L the survival rate of the test animals at the end of the test was 90 - 100 %. At the two highest test concentrations of nominal 6.3 and 20 mg test substance/L the survival rate of the test animals had decreased to 70 % and 80 %, respectively. Thus, the survival rate of Daphnia magna after 21 days was significantly (>20 %) reduced first at the test concentration of nominal 6.3 mg test substance/L (corresponding to a mean test substance concentration of 4.9 mg/L).

Reproduction rate:
The mean reproduction rate of those Daphnia in the control was 89.3 ±15.2 (mean ± SD) alive offspring per adult. According to the results of a Williams-test (one-sided, a = 0.05) no significant toxic effect of the test substance on the mean reproduction rate was determined up to and including the test concentration of nominal 0.63 mg test substance/I (mean measured concentration of 0.49 mg/l). First at the nominal concentration of 2.0 mg test substance/I (mean measured 1.55 mg/l), the mean reproduction rate of surviving Daphnia was statistically significant reduced to in the mean 79.5 % alive offspring compared to the control. With increasing test substance concentrations the mean reproduction rate further decreased.

Signs of intoxication:
With exception of the reported mortality and the reduced reproduction rates, no particular signs of intoxication were observed at the test animals during the test.

Reported statistics and error estimates:

The NOEC and the LOEC of the reproduction rate were statistically evaluated by the multivariate Williams-test after a one-way analysis of variance (ANOVA). Additionally, the EC10 and EC50 of the reproduction rate after 21 days was statistically evaluated by Probit analysis. All biological results respectively the calculations were based on the mean measured test substance concentrations.

If the quantification of the test substance in the aged samples is based on the sum of main compound and degradation product, 67 - 76 % of nominal were found in the samples from the test media without food particles, respectively 42 - 68 % of nominal in the samples out of the real test media with food particles and test animals. Thus, a part of the degradation product had obviously adsorbed onto the food particles. However, since filter feeding test animals as Daphnia can take up the test substance also from ingested food, the decrease of test substance concentration in water due to adsorption onto food is not taken into account. Therefore, the biological results are based on the mean measured test substance concentrations in the test media without food. Since a toxic effect can be caused by the main compound as well as by the degradation product, the measured concentrations of main compound plus degradation product are taken into account. At the nominal test concentration of 0.63 mg/l this mean measured concentration amounted to 0.49 mg/l (77 % of nominal), at nominal 2.0 mg/l to 1.55 mg/l (78 % of nominal).

Validity criteria fulfilled:

yes

Conclusions:

The 21-day EC10 and EC50-values for the reproduction rate of the test animals were calculated by Probit analysis (based on mean test substance concentrations). The 21-day EC10 amounted to 0.8 mg/L with a 95 % confidence interval from 0.03 to 2.1 mg/L, the 21- day EC50 amounted to 8.6 mg/L with a 95 % confidence interval from 3.3 to 158 mg/L.

Executive summary:

The influence of the test substance FAT 40549/A on the reproduction and survival rate of Daphnia magna was investigated in a 21 days semi-static test following the OECD Guideline for Testing of Chemicals, Proposal for updated Guideline 211, Revised Draft Document December 1996: "Daphnia magna Reproduction Test".

The nominal test concentrations were 0.20, 0.63, 2.0, 6.3 and 20 mg test substance/L and a control.

In the test media the test substance degraded into a defined reaction product. The mean measured concentrations in the samples from the freshly prepared test media ranged from 76 % to 90 % of the nominal concentrations, if the quantification is based on the main compound. If the quantification is based on the sum of main compound and degradation product, the mean recoveries in the non-aged samples were nearly identical. Therefore, the biological results are based on the mean measured test substance concentrations in the test media without food. Since a toxic effect can be caused by the main compound as well as by the degradation product, the measured concentrations of main compound plus degradation product are taken into account.

Taking into account the survival rates and the reproduction rates of the test animals, the highest concentration of FAT 40549/A tested without toxic effects after the exposure period of 21 days (21-day NOEC) was determined to be at least 0.8 mg/L, since at this concentration the test substance respectively its degradation product affected the mean reproduction rate of the test animals by not more than 10%, and the survival rate was not significantly reduced. The lowest concentration tested with toxic effects (21-day LOEC) was determined to be 1.55 mg/L due to the statistically significant reduced mean reproduction rate of Daphnia magna at this mean measured test substance concentration. The survival rate of the adult Daphnia was reduced first at the mean test concentration of 4.9 mg/L (nominal 6.3 mg test substance/L).

The 21-day EC10 and EC50-values for the reproduction rate of the test animals were calculated by Probit analysis (based on mean test substance concentrations).

The 21-day EC10 amounted to 0.8 mg/L with a 95 % confidence interval from 0.03 to 2.1 mg/L, the 21 - day EC50 amounted to 8.6 mg/L with a 95 % confidence interval from 3.3 to 158 mg/L.

Description of key information

The 21-day NOEC was determined to be at least 0.8 mg/L, and the 21-day LOEC was determined to be 1.55 mg/L.

In the OECD 211 study the test substance Reactive yellow 205 was found degraded into a defined reaction product in the test media. The decrease of the concentrations during the test medium renewal periods of 48 and 72 hours was obviously caused by degradation of the main compound due to hydrolysis. The degradation of test substance was noted in the 48 hr test media sample, and there was no mortality observed during this time (48 hr) at the highest test concentration of 20 mg/l. Hence, the toxicity observed (reproduction rate) could be due to the degraded product or could be a synergic effect of test substance and degraded product. There is no separate tox data for test substance and the degraded product to confirm this.

Therefore, based on the analytical evidence that the test substance degraded in the test medium its proposed not to classify this substance based on chronic test data as the observed results could not be corelated only to the the test substance (Reactive Yellow 205).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.8 mg/L

Additional information

The influence of the test substance FAT 40549/A on the reproduction and survival rate of Daphnia magna was investigated in a 21 days semi-static test following the OECD Guideline for Testing of Chemicals, Proposal for updated Guideline 211, Revised Draft Document December 1996: "Daphnia magna Reproduction Test".

 

The nominal test concentrations were 0.20, 0.63, 2.0, 6.3 and 20 mg test substance/L and a control.

 

In the test media the test substance degraded into a defined reaction product. The mean measured concentrations in the samples from the freshly prepared test media ranged from 76 % to 90 % of the nominal concentrations, if the quantification is based on the main compound. If the quantification is based on the sum of main compound and degradation product, the mean recoveries in the non-aged samples were nearly identical. Therefore, the biological results are based on the mean measured test substance concentrations in the test media without food. Since a toxic effect can be caused by the main compound as well as by the degradation product, the measured concentrations of main compound plus degradation product are taken into account.

 

Taking into account the survival rates and the reproduction rates of the test animals, the highest concentration of FAT 40549/A tested without toxic effects after the exposure period of 21 days (21-day NOEC) was determined to be at least 0.8 mg/L, since at this concentration the test substance respectively its degradation product affected the mean reproduction rate of the test animals by not more than 10%, and the survival rate was not significantly reduced. The lowest concentration tested with toxic effects (21-day LOEC) was determined to be 1.55 mg/L due to the statistically significant reduced mean reproduction rate of Daphnia magna at this mean measured test substance concentration. The survival rate of the adult Daphnia was reduced first at the mean test concentration of 4.9 mg/L (nominal 6.3 mg test substance/L).

 

The 21-day EC10 and EC50-values for the reproduction rate of the test animals were calculated by Probit analysis (based on mean test substance concentrations).

The 21-day EC10 amounted to 0.8 mg/L with a 95 % confidence interval from 0.03 to 2.1 mg/L, the 21 - day EC50 amounted to 8.6 mg/L with a 95 % confidence interval from 3.3 to 158 mg/L.