trisodium 3-amino-4-[4-[4-(2-(2-ethenylsulfonylethoxy)ethylamino)-6-fluoro-1,3,5-triazine-2-ylamino]-2-sulfophenylazo]-5-hydroxynaphthalene-2,7-disulfonate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment start date - 19 March 1999; Experiment completion date - 22 May 1999; Study completion date - 22 June 1999.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identity: FAT 40'574/B
Batch: WP 23/99
Purity: Approx. 75 %
Appearance: Solid, dark-red powder
Storage: At room temperature at about 20 °C
Expiration Date: 08 February 2006

Analytical monitoring:

yes

Details on sampling:

For the analytical measurements of the test item concentrations, duplicate samples were taken at the start of the test from the freshly prepared test media (without algae) of all test concentrations and from the control. For the determination of the stability of the test item under the test conditions, and for the maintenance of the test item concentrations during the test period respectively, sufficient volumes of the freshly prepared test media of all test concentrations and the control were incubated under the same conditions as in the actual test (but without algae), and were sampled in duplicate at the end of the test (after the 72 hours test period). All samples were deep-frozen (at about -20 °C) immediately after sampling analyses (the test item was sufficiently stable in the test media under the storage conditions as determined in a pre-experiment). The concentrations of the test item FAT 40574/B were measured in the duplicate test media samples from all test concentrations from both sampling times (0 and 72 hours). From the control samples only one of the duplicate samples was analyzed from each of both sampling times (0 and 72 hours). Remaining samples are kept stored at about -20 °C to enable additional.

Vehicle:

no

Details on test solutions:

Identity and concentration of auxiliary solvent for dispersal: None

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test organism used for the study was Scenedesmus subspicatus CHODAT, Strain No. 86.81 SAG, supplied by the "Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen", D-37073 Göttingen. The algae had been grown in the RCC laboratories under standardized conditions according to the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

22 °C

pH:

7.8 - 8.1

Nominal and measured concentrations:

Nominal : 0 (control) 1.0, 3.2, 10, 32, 100 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed, Each Erlenmeyer flask was placed in a black cylinder, coated inside with aluminum foil. The cylinders were covered with glass dishes, the dishes were covered with watch glass dishes to prevent evaporation
- Material, size: Glass, 50 mL.
- Aeration: No
- Initial cells density: 10000 algal cells per ml
- No o flask per concentration - 3 flasks per test concentration and 6 flasks in the control. 3 replicates per test concentration.

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination at a mean light intensity of about 7200 Lux, range 6800 - 7900 Lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: Counting chamber

TEST CONCENTRATIONS
- Range finding study: No

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Basis for effect:

biomass

Details on results:

Analytical results:
The analytically determined mean test item concentrations in the analyzed test media varied in the range from 96 to 109 % of the nominal values (see the analytical results. The test item FAT 40574/B was sufficiently stable in the test media under the test conditions during the test period of 72 hours. Therefore, all biological results are related to the nominal concentrations of the test item.

Biological results:

Experimental part A: Experimental part A corresponds to the usual algal toxicity test. This means that the algal growth inhibition in this experimental part was caused by a possible toxic effect of the test item and/or by the reduced light intensities due to the light absorption in the colored test media. In experimental part A, the test item had a statistically significant inhibitory effect on the mean growth rate u and the mean biomass b of Scenedesmus subspicatus after the exposure period of 72 hours first at the nominal concentration of 10 and 3.2 mg/l, respectively. From this results, the 72-hour LOEC (lowest concentration tested with toxic effects) was determined to be the nominal concentration of 3.2 mg/l. The 72-hour NOEC (highest concentration tested without toxic effects after a test period of 72 hours) was determined at the concentration of 1.0 mg/l, since up to and including this test concentration the mean biomass b of the algae was statistically not significantly lower than in the control. The mean growth rate µ was statistically not significantly reduced even at 3.2 mg/l. At the microscopical examination of the shape of the algal cells after 72 hours incubation period no difference was observed between the algae growing in the test item concentration of 10 mg/l in experimental part A and the algal cells in the control. Thus, the shape of the algal cells, growing at this concentration of dissolved test item was obviously not affected.

Experiment part A

Parameter	Biomass b (mg/l)	Growth rate µ (mg/l)
EC50	32	156
EC10	1.9	10

 

Experimental part B

In experimental part B the algal growth inhibition caused by the pure light effect (the reduced light intensities in the colored test media) was quantified. In this experimental part a similar inhibition effect on the algal growth was observed compared to experimental part A. In experimental part B both, the mean growth rate µ and the mean biomass b of Scenedesmus subspicatus, was significantly reduced compared to the control after 72 hours test period first at the test concentration of 32 mg/l. The EC values in experimental part B are listed below:

Parameter	Biomass b (mg/l)	Growth rate µ (mg/l)
EC50	67	190
EC10	9.3	22

 

Comparison between the results in experimental parts A and B

According to the recommendations of the Ad-hoc working group of experts on algal growth inhibition for the interpretation of test results of colored substances, the comparison between the results in experimental parts A and B was based on the growth rates. The differences between the results of experimental parts A and B were described for each test concentration as percentage inhibition of the growth rate µA (lMA) minus the percentage inhibition of the growth rate µB (IMB) after the 72 hours test period. At all test concentrations these differences were lower than 10 %. As another measure of difference the quotient of the growth rates µA/µB was calculated for each test concentration. At all test concentrations this quotient was at least 0.9 or higher. Differences in growth rates up to the magnitude of 10 % are accepted to be caused by pure chance in the used algal toxicity test. Thus, according to the recommendations of the Ad-hoc working group of experts on algal growth inhibition tests for colored substances the differences between inhibition in experimental part A and B should be not higher than 10 %, respectively the quotient µA/µB should be at least 0.9 or higher to accept that the inhibition curves of the growth rates µA and µB are essentially the same. At all test concentrations of this test the differences of the growth rates µA and µB are lower than 10 % and the quotients µA/ µB are at least 0.9.

Validity criteria fulfilled:

yes

Conclusions:

The modified algal test has clearly demonstrated that the observed growth inhibition effect of the test item on Scenedesmus subspicatus was caused only due to an indirect effect, the light filter effect in the colored test solutions. Thus, a toxic effect of the test item on the algal cells can be excluded up to the highest test concentration of nominal 100 mg/l. According to Directive 93/21 EEC (adaption to Directive 67/548/EEC), the ecotoxicological classification is non-toxic algal cells after the test period 72 h.

Executive summary:

The influence of the test item FAT 40574/B on the growth of the green algal species Scenedesmus subspicatus CHODAT was investigated in a 72-hour static test according to the Commission Directive 92/69/EEC, Annex Part C.3, 1992, and the OECD Guideline No. 201, 1984. However, the test method was modified to quantify the algicidal effect of the test item, but also the growth inhibition effect caused by reduced light intensities in the colored test solutions. The nominal test concentrations were 1.0, 3.2, 10, 32, 100 mg/l and a control. All test media down to the lowest test concentration were slightly to strongly colored by the test item. The analytically determined mean test item concentrations in the analyzed test media varied in the range from 96 to 109 % of the nominal values. The test item FAT 40574/B was sufficiently stable in the test media under the test conditions during the test period of 72 hours. Therefore, all biological results are related to the nominal concentrations of the test item. The same growth inhibition of Scenedesmus subspicatus was observed when the algae grew in test water without test item, but under reduced light intensities by the filter effect of the colored test media as in the second parallel experimental part, where the algae grew in the test media with dissolved test item. Thus, in conclusion, this modified algal test has clearly demonstrated that the observed growth inhibition effect of the test item FAT 40574/B on Scenedesmus subspicatus was caused only due to an indirect effect, the light filter effect in the colored test solutions. Thus, a toxic effect of the test item on the algal cells can be excluded up to the highest test concentration of nominal 100 mg/l.

Description of key information

The EC50 growth rate was determined to be greater than 100 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The influence of the test item FAT 40574/B on the growth of the green algal species Scenedesmus subspicatus Chodat was investigated in a 72-hour static test according to the Commission Directive 92/69/EEC, Annex Part C.3, 1992, and the OECD Guideline No. 201, 1984. However, the test method was modified to quantify the algicidal effect of the test item, but also the growth inhibition effect caused by reduced light intensities in the colored test solutions. The nominal test concentrations were 1.0, 3.2, 10, 32, 100 mg/l and a control. All test media down to the lowest test concentration were slightly to strongly colored by the test item. The analytically determined mean test item concentrations in the analyzed test media varied in the range from 96 to 109 % of the nominal values. The test item FAT 40574/B was sufficiently stable in the test media under the test conditions during the test period of 72 hours. Therefore, all biological results are related to the nominal concentrations of the test item. The same growth inhibition of Scenedesmus subspicatus was observed when the algae grew in test water without test item, but under reduced light intensities by the filter effect of the colored test media as in the second parallel experimental part, where the algae grew in the test media with dissolved test item. Thus, in conclusion, this modified algal test has clearly demonstrated that the observed growth inhibition effect of the test item FAT 40574/B on Scenedesmus subspicatus was caused only due to an indirect effect, the light filter effect in the colored test solutions. Thus, a toxic effect of the test item on the algal cells can be excluded up to the highest test concentration of nominal 100 mg/l.