C.I. Reactive Yellow 161

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:

Feb - Mar 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

1992

Principles of method if other than guideline:

The test method was modified following a method developed by RCC Ltd. to quantify the algicidal effect of colored test substances, but also the growth inhibition effect caused by reduced light intensities in the colored test media.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For the analytical measurements of the test article concentrations, duplicate samples were taken at the start and end of the test from freshly prepared test media (without algae) of all test concentrations and from control.

Vehicle:

no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Scenedesmus subspicatus Strain No.: 86.81 SAG supplied by "Sammlung von Algenkulturen, Pflanzenphysiologisches institut" Gottingen University. The algae had been grown in the RCC laboratories under standardized conditions according to the test guidelines.

Test type:

static

Water media type:

freshwater

Remarks:

synthetic test water prepared according to the mentioned guidelines

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (24 mg/L) as CaCO3

Test temperature:

23 -2 4°C

pH:

7.9 - 9.2

Nominal and measured concentrations:

Nominal: 0 (control), 1.0, 3.2, 10, 32, 100 mg/L
Measured (mean): 8.5, 27 78 mg/L. 0 (control), 1.0 and 3.2 mg/L were not measured

Details on test conditions:

The test was started by inoculation of a biomass of 10.000 algal cells per ml test medium. These cells were taken from an exponentially growing pre-culture, which was set up three days prior the test.
The test was performed in Erlenmeyer flasks (50 ml), each with 15 ml algal suspension, continuously stirred by magnetic stirrers, 3 flasks per test concentration and 6 flasks in the control. Each Erlenmeyer flask was placed in a black cylinder, coated inside by aluminum foil. The cylinders were covered with glass dishes, the dishes were covered with watch glass dishes to prevent evaporation.

The test included two experimental parts:
A:
The algae grew in test media with dissolved test substance in the Erlenmeyer flasks (five test concentrations and a control). All glass dishes above cylinders contained purified water. Thus, the inhibition of algal growth in this part was caused due to real toxic effect of the test substance and in addition to reduced light intensities in the colored test media in the Erlenmeyer flasks.
B:
In this part glass dishes above the cylinders contained the colored test media with the same concentrations as in part A, however without algae (3 replicates per test concentration). In the Erlenmeyer flasks below, the algae grew in the water without test substance (as in control), however under changed light conditions due to the filter effect of the colored test media in the glass dishes. Thus the growth inhibition in part B was caused due to light absorption only. The depth of the test media in the glass dishes was 4 mm – half of the depth of the media in Erlenmeyer flasks. All flasks were incubated in a temperature controlled water bath and continuously illuminated at a mean light intensity of about 7500 Lux (fluorescent tubes).

Counting and Examination of the algal cells:
Small volume of the test media (1.0-2.0 mL) were taken out of all test flasks after 2, 48 and 72 hours of exposure and were not replaced. The algae cell densities in the samples were determined by counting with an electronic particle counter (Coulter Counter, Model ZM), two measurements per sample. In addition, a sample was taken from the control and from the test concentration of nominal 46 mg test article/L in experiment A with reduced algal growth after the period of 72 hours. The shape of these algal cells was microscopically examined and compared with the cells in the control.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 78 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

35 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

79 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

9.9 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Experiment A:
In experimental part A, the test item had a statistically significant inhibitory effect on the growth rate r of Scenedesmus subspicatus after the exposure period of 72 hours first at the mean measured concentration of 78 mg/L (results of a Dunnett-test, one-sided, a = 0.05). Thus, this test concentration was determined as the 72-hour LOEC (lowest concentration tested with toxic effects). The 72-hour NOEC (highest concentration tested without toxic effects after a test period of 72 hours) was determined at the mean measured concentration of 27 mg/L, since up to and including this test concentration the mean growth rate r of the algae was statistically not significantly lower than in the control. For the biomass b, the 72-hour LOEC and the 72-hour NOEC were determined to be at the mean measured concentrations of 27 mg/L and 8.5 mg/L, respectively.

Experiment 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 only a slightly lower inhibition effect on the algal growth was observed compared to experimental part A. Thus, a great part of the algal growth inhibition was obviously caused by the pure light effect. The EC values in experimental part B were slightly higher than the corresponding EC values
in experimental part A.

Comparison 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. At the lower test concentrations these differences were lower than 10%. However, at the highest test concentration of 100mg/L the difference was higher than 10%. As another measure of difference, the quotient of the growth rates rA/rB 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%, and the quotient rA/rB should be at least 0.9 or higher to accept that the inhibition curves of
the growth rates rA and rB are essentially the same. At the highest test concentration of this test, the difference of the inhibition of growth rates rA
and rB is higher than 10% while the quotients rA/rB are at least 0.9. Thus, a real toxic effect of the test item on the growth of Scenedesmus subspicatus can not be excluded at the test concentration of nominal 100 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

The growth rate and biomass growth were determined as follows, in experiment A, growth rate: EC10: 35 mg/L; EC50: >78 mg/L; biomass: EC10: 9.9 mg/L; EC50: 79 mg/L. In experiment B, growth rate: EC10: >78 mg/L; EC50: >78 mg/L; biomass: EC10: 38 mg/L; EC50: >78 mg/L. The substance is not acutely or chronically toxic to freshwater green algae. Slight inhibitoty effects observed during this test were mainly indirect due to light absorption (algistatic). The substance has only low potential for an algicidal effect. The substance is not classifiable as all relevant ErC10 and ErC50 values are higher than the cut-off criteria specified in Regulation 1272/2008. The EC50 of >78 mg/L (measured) corresponds to a nominal value of >100 mg/L.

Executive summary:

The influence of the test item on the growth of the green algal species Scenedesmus subspicatus CHODAT was investigated in a 72-hour static test according to the EU 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 and 100 mg/L in parallel with a control. All test media down to the lowest test concentration were slightly to strongly colored by the test item. The measured test item concentration in the analyzed test media samples (10, 32 and 100 mg/L) amounted to 84, 82 and 76% of the nominal values at the start of the test. In these test media, incubated under the conditions of the test during the test period (but without algae), the measured concentrations amounted to 102-106% of the initially measured values after 72 hours. Consequently, the test item was stable during the test period of 72 hours under the conditions of the test. The mean measured test concentrations (calculated as the average over all measurements per test concentration) varied in the range of 78 to 85% of the nominal values. The reported biological results are based on the mean measured test item concentrations of 8.5 mg/L (nominal 10 mg/L), 27 mg/L (nominal 32 mg/L) and 78 mg/L (nominal 100 mg/L). The two lowest test concentrations (nominal 1.0 and 3.2 mg/L) were not analyzed since they were below the 72-hour NOEC. The growth inhibition effect on Scenedesmus subspicatus was caused in part by the indirect effect, the light absorption in the colored test solutions. However, at the highest test concentration a real toxic effect of the test item on the growth of Scenedesmus subspicatus could not be excluded. Therefore, the results of this experimental part, where the algae grew in the test media with dissolved test item as in a usual algal growth inhibition test should be taken into consideration for the determination of the toxic effect of the test item on the growth of Scenedesmus subspicatus. In this experimental part the test it had a statistically significant inhibition effect on the growth rate of Scenedesmus subspicatus after the exposure period of 72 hours first at the mean

measured concentration of 78 mg/L (= 72-hour LOEC: lowest concentration tested with toxic effects). The 72-hour NOEC (highest concentration tested without toxic effects) was determined at the mean measured concentration of 27 mg/L, since at this test concentration the mean growth rate r of the algae was statistically not significant lower than in the control. For the biomass b, the 72-hour LOEC and the 72-hour NOEC were determined to be at the mean measured concentrations of 27 mg/L and 8.5 mg/L, respectively.

Description of key information

The growth rate and biomass growth were determined as follows, in experiment A, growth rate: EC10: 35 mg/L; EC50: >78 mg/L; biomass: EC10: 9.9 mg/L; EC50: 79 mg/L. In experiment B, growth rate: EC10: >78 mg/L; EC50: >78 mg/L; biomass: EC10: 38 mg/L; EC50: >78 mg/L. The substance is not acutely or chronically toxic to freshwater green algae. Slight inhibitoty effects observed during this test were mainly indirect due to light absorption (algistatic). The substance has only low potential for an algicidal effect. The substance is not classifiable as all relevant ErC10 and ErC50 values are higher than the cut-off criteria specified in Regulation 1272/2008. The ErC50 of >78 mg/L (measured) corresponds to a nominal value of >100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

35 mg/L