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Diss Factsheets

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 - 18 Oct 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
28 Jul 2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
24 Aug 2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany (11 Apr 2011)

Test material

Constituent 1
Chemical structure
Reference substance name:
1-Naphthalenesulfonic acid, 6-diazo-5,6-dihydro-5-oxo-, ester with phenyl(2,3,4-trihydroxyphenyl)methanone
EC Number:
270-931-7
EC Name:
1-Naphthalenesulfonic acid, 6-diazo-5,6-dihydro-5-oxo-, ester with phenyl(2,3,4-trihydroxyphenyl)methanone
Cas Number:
68510-93-0
Molecular formula:
all potential esters of C10HH5O3N2SCl and C13H10O4
IUPAC Name:
3-benzoyl-2-hydroxy-6-({[5-oxo-6-(λ⁵-diazynylidene)-5,6-dihydronaphthalen-1-yl]sulfonyl}oxy)phenyl 5-oxo-6-(λ⁵-diazynylidene)-5,6-dihydronaphthalene-1-sulfonate; 4-benzoyl-2,3-bis({[5-oxo-6-(λ⁵-diazynylidene)-5,6-dihydronaphthalen-1-yl]sulfonyl}oxy)phenyl 5-oxo-6-(λ⁵-diazynylidene)-5,6-dihydronaphthalene-1-sulfonate; 4-benzoyl-2,3-dihydroxyphenyl 5-oxo-6-(λ⁵-diazynylidene)-5,6-dihydronaphthalene-1-sulfonate
Test material form:
solid

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control, 0.1, 0.32, 1.0, 3.2, 10, 32, and 100 mg/L (nominal)
- Sampling method: For each treatment 200 mL of the respective test item solution was mixed with the necessary amount of algal pre-culture. In this mixture, the pH-value was measured and samples for the analytical determination were taken.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A water-accommodated fraction of the load 100.1 mg/L was prepared for the test. This was done by mixing the nominal load with the corresponding amount of nutrient medium (demineralised water enriched with minerals but without algae) and shaking vigorously for 24 h. The resulting solution was filtered through 0.45 µm Nylon filters. The lower treatments were prepared by dilution of this solution with nutrient medium.
- Controls: Deionised water with nutrient medium and alga.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: freshwater green microalgae
- Strain: SAG 86.81
- Source: MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen, Germany), Feb 2014.
- Age of inoculum (at test initiation): 4 d
- Method of cultivation: The algae are kept as stock culture on solid agar at 7 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared. Four days before the start of the test, an aliquot of the permananent culture was brought into pre-culture medium and incubated under continuous lighting for 96 h. The resulting culture is growing exponentially.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
21.5 - 23.4 °C
pH:
0 h: 7.6 - 7.7
72 h: 7.8
Nominal and measured concentrations:
Control, 0.1, 0.32, 1.0, 3.2, 10, 32, and 100 mg/L (nominal)
3.25/2.39, 1.29/0.22, 0.47/0.63, 0.54/0.00, 0.02/0.40, 0.76/0.63, 2.15/2.29, 7.79/7.83 mg/L DOC (measured 0 h/ 72 h)
Details on test conditions:
TEST SYSTEM
- Test vessel: flasks
- Material, size, headspace, fill volume: 65 mL glass flasks filled with 45 ± 1 mL test solution.
- Initial cells density: 2.156E+03 cells/mL
- Control end cells density: 1,55232E+05 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes, nutrient medium (enriched with minerals)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: Same as test.
- Intervals of water quality measurement: The pH-values were measured at test start and end. The DOC content in the test vessels was measured at the start and end of the test. Temperature was measured with a data logger for temperature (Ebro).

OTHER TEST CONDITIONS
- Sterile test conditions: Yes, all solutions were sterilised before use.
- Photoperiod: continuous
- Light intensity and quality: 5800 Lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: spectrophotometer: after 24, 48, and 72 h
- Other: Microscopic assessment of cell aspect: at the end of the test

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
12 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accommodated Fraction (WAF)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
8.1 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accommodated Fraction (WAF)
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): Yes, cell number increased by a factor 72 in 72 h (required: factor 16 in 72 h).
- Observation of abnormalities (for algal test): No
Results with reference substance (positive control):
- Results with reference substance valid? Yes, last GLP study with reference substance dating Jul 2014.
- ErC50 (72 h) = 0.99 mg/L potassium dichromate (95% confidence interval: 0.94 - 1.1 mg/L).
Reported statistics and error estimates:
For the treatment with the test item concentration 0.1 mg/L, it was tested whether the differences between treatment and control were significant. The values for the yield and for the growth rate were used for the assessment of observed effect levels.
In order to select a suitable test for significance, it was checked whether equality of variance was given. The calculated value F was compared with the F-test table (level of significance 95%). If the calculated value was smaller than the tabular value, equality of variance was given.
As equality of variance was given, the t-test was used.
With the t-test, it was checked whether the differences are significnat. Significance is given if the calculated t-value is bigger than the limit of significance (t-value taken from the table with grade of freedom: n1 + n2-2, level of significance 95%).
For the endpoints growth rate and yield, the differences between the nominal concentration 0.1 mg/L and the control can be considered as significant (level of significance: 97.5%) as the calculated t-values were higher than the limit of significance.
The estimation of the EC50s and EC10s of the test item was accomplished with the software Origin. The data were evaluated with a sigmoidal fit.

Any other information on results incl. tables

ANALYTICAL RESULTS

At the start and at the end of the test, the content of dissolved organic carbon in the test solutions was determined using a carbon analyser.

Dissolved organic carbon (DOC) was calculated by subtraction of the inorganic carbon values (IC) from the total organic carbon values (TC).

The detected concentrations of dissolved organic carbon were in the same range in all treatments (control and treatments) except for the highest treatment. Only in the highest treatment, the presence of dissolved test item was observable (Table 1). Therefore, the determination of the results was based on the nominal concentrations.

Table 1. Measured DOC concentrations.

Nominal concentration

test item

Measured DOC

t = 0 h

Measured DOC

t = 72 h

mg/L

mg/L

mg/L

Blank control

3.25

2.39

0.1

1.29

0.22

0.32

0.47

0.63

1.0

0.54

0.00

3.2

0.02

0.40

10

0.76

0.63

32

2.15

2.29

100

7.79

7.83

 

BIOLOGICAL RESULTS

Because the test item is light sensitive, it is not determinable whether toxicity was caused by the test item or by its metabolites. Degradation resp. transformation under test conditions was clearly observed by a change of colour in the higher concentrated test solutions. At the end of the test, absorption in the two highest treatments was lower than at the start of the test. In the microscopic observation, no algal cells could be detected in these treatments. Therefore, their inhibition was stated as 100%. As decolouration of the test item also occurred in the lower treatments, the increase of the measured absorption values at the end of the test might be reduced by decolouration. That means that the calculated inhibition values based on photometric measuremet might be slightly increased. Therefore, the result of the test is regarded as worst-case scenario.

In the two highest treatments, no algal cells were visible at the end of the test and absorption at the end of the test was lower than at the start of the test. Therefore, inhibition of algal growth was stated as 100% for these treatments. In the third highest treatment (10 mg/L nominal concentration), no cells were visible in the microscopical observation at the end of the test. As a slight increase of absorption at the end of the test compared to the absorption at the start of the test was observed, a slight growth of algae was estimated. Therefore, the detected absorptions were used for determination of cell numbers and evaluation of inhibition values.

Table 2. Mean inhibition Values.

Nominal concentration

[mg/L]

% Inhibition

Growth rate

(0 - 72 h)

Yield

(0 – 72 h)

Blank control

0

0

0.1

3.10

12.44

0.32

3.97

15.73

1.0

3.01

12.21

3.2

6.62

24.65

10

24.31

64.79

32

100.00

100.00

100

100.00

100.00

 

MICROSCOPICAL OBSERVATIONS

After 72 h the test solutions with a nominal concentration of 10, 32, and 100 mg/L appeared yellowish-green. In all other test solutions the alga appeared normal and healthy.

Applicant's summary and conclusion