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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 03-Dec-2018 to 07-Dec-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
yes
Remarks:
uncritical
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2015
Deviations:
yes
Remarks:
uncritical
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Because the test item was not completely soluble in algal medium, a saturated solution was prepared for the test. This was done by mixing the nominal load of 10 mg (real load see table below) with the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and shaking vigorously for 24 ± 1 hours. The resulting solution was filtrated through 0.45 µm PTFE filters.
The lower treatments (5.6 / 3.2 / 1.8 / 1.0 mg/L nominal) were prepared by dilution of this solution with algal medium.
Sample preparation
Fivefold Enrichment (treatments 10 mg/L and 5.6 mg/L)
4 g NaCl were added to 50 mL of the test item solution in matrix; then, the clear solution was extracted two times with the solvent ethyl acetate (8 and 4 mL), the organic phase was collected into a 10 mL flask, after drying with Na2SO4. The flask was filled up to 10 mL with ethyl acetate after addition of 500 µL ISTD (internal standard) stock solution (1000 mg/L in ethyl acetate) and the solution was measured via GC/FID.
Tenfold Enrichment (treatment 3.2 mg/L)
6 g NaCl were added to 100 mL of the test item solution in matrix; then, the clear solution was extracted two times with the solvent ethyl acetate (10 and 4 mL), the organic phase was collected into a 10 mL flask, after drying with Na2SO4. The flask was filled up to 10 mL with ethyl acetate after addition of 500 µL ISTD (internal standard) stock solution (1000 mg/L in ethyl acetate) and the solution was measured via GC/FID.
Fiftyfold Enrichment (treatments 1.8 and 1 mg/L)
6 g NaCl were added to 100 mL of the test item solution in matrix; then, the clear solution was extracted two times with the solvent ethyl acetate (10 and 4 mL), the organic phase was collected into a flask after drying with Na2SO4, the most of the solvent ethyl acetate was evaporated and the residue was quantitatively transferred into a 2 mL flask. The flask was filled up to 2 mL with ethyl acetate after addition of 100 µL ISTD (internal standard) stock solution (1000 mg/L in ethyl acetate) and the solution was measured via GC/FID.

Miscellaneous
On each sampling day, validity of calibration was controlled by measuring QC samples.
At the measurement of 0 h and 24 h, the deviation of the QC sample was higher than ± 3 % and lower than ± 15 %. Therefore, the recovery rate calculated from the QC samples was taken into account as sensitivity correction factor using the following equations:

QC Recovery in % = (measured concentration/nominal concentration)*100%

At the measurement of 48 h, the deviation of the QC sample was higher than ± 15 % and therefore a new calibration was performed without level 3 mg/L, because the recovery rate lay outside 100 ± 20 %.
The recovery rate calculated from the QC samples was taken into account at the meas-urement of 48 h and 72 h using the equations above. The deviations of the means of these samples were higher than ± 3 % and lower than ± 15 %.
Furthermore, the recovery rate of the medium was taken into account at each measure-ment.
Vehicle:
no
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Selection of the test system was made following the proposal of the guidelines.
Specification
Unicellular freshwater green alga.
Genus Desmodesmus
Species subspicatus
SAG Strain Number 86.81
Taxonomic position Chlorophyta - Chlorophyceae
Origin and Culture
The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sci-encebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21.7 – 22.8 °C
pH:
8.1 - 9.7
Nominal and measured concentrations:
At the beginning of the test, there is a high correlation between nominal and measured concentration: the measured concentrations lay between 87 % and 100 % of the nominal concentrations. But due to insufficient stability of the test item, the measured concentra-tions lay between 51 % and 73 % of the nominal concentrations after 24 h, between 21 % and 40 % of the nominal concentrations after 48 h and between 0 % and 12 % at the end of the test.
In addition, the scattering values at the end of the test, might be caused by the presence of the algae cells (adsorption or ingestion of dissolved test item onto the algae cells). That means a part of the test item was present but not measurable. This effect can be exclud-ed in the highest concentration due to nearly complete inhibition of algae growth.
Therefore, the determination of the results was based on the calculated geometric mean of the measured concentrations in the highest concentration and the respective dilution factor (calculated mean).
Measured Concentrations
0 h 24 h 48 h 72 h
Nominal Concentration Measured Concentration Test Item
mg/L mg/L mg/L mg/L mg/L
1 0.87 0.73 0.40 0.12
1.8 1.80 1.19 0.47 0.14
3.2 3.03 1.62 0.68 0.10*
5.6 5.12 3.49 2.19 0.10*
10 9.70 6.24 3.18 1.09
LOD 0.10 0.10 0.10 0.10

n. a. = not analysable
LOD (limit of detection) was determined using the lowest concentration of the calibration and taking the dilution factor into account.
* At the concentrations 3.2 mg/L and 5.6 mg/L, no test item could be measured after 72 h. Therefore, the LOD was used for the determination of the geometric mean.

Geometric and Calculated Geometric Means of the Measured Concentrations
Nominal Concentration Test Item Geometric Mean Calculated Geometric Mean*
1 0.42 0.38
1.8 0.61 0.68
3.2 1.49 1.22
5.6 3.39 2.13
10 3.80 3.80
* Calculated considering the dilution factors for each nominal concentrations and the geometric mean of the highest nominal concentration.
Details on test conditions:
Treatments tested: 1 / 1.8 / 3.2 / 5.6 / 10 mg/L nominal concentration
The concentrations to be tested are based on non GLP pre-tests
Number of replicates: 6 replicates for the blank control, each completely filled with algal medium and algae
3 replicates for each treatment, each completely filled with test solution and algae
Vessels: closed glass flasks total volume 65 mL
Duration: 72 hours
Lighting: 5000 lux
Temperature: 21.7 – 22.8 °C
Reference substance (positive control):
yes
Remarks:
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate reference test.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.22 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.22 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
2.13 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
5.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
2.13 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
5.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.75 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: C.I. 1.34 mg/L – 2.29 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.02 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: C.I. 0.59 mg/L – 1.32 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 3.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.33 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: C.I. 1.96 mg/L – 2.80 mg/L
Results with reference substance (positive control):
Biological Results of the Reference Study (201803R301)
The results of the last study with the positive control K2Cr2O7 are presented in the follow-ing table. The study was performed under GLP conditions in January 2018.
Table 6.2 a Biological Results of K2Cr2O7
Parameter Value 95% confidence interval
72h ErC50 0.79 mg/L 0.79 mg/L – 0.79 mg/L
72h EyC50 0.39 mg/L 0.35 mg/L – 0.45 mg/L
Reported statistics and error estimates:
Calculation of results was performed with the help of validated software (Microsoft Ex-cel®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.2.1. The details of calculation are stated in Annex 6: Statistical cal-culation using ToxRat® Professional 3.2.1.
Validity criteria fulfilled:
yes
Remarks:
Increase factor biomass=95, Mean coefficient of variation of daily growth rates=24%, Coefficient of variation of average growth rate during the whole test period=2%
Conclusions:
ErC50 (72 h) >3.80 mg/l
EyC50 (72 h) =2.33 mg/l
Executive summary:

One valid experiment was performed.

The study was performed using 5 concentrations ranging from 1.0 to 10 mg/L (nominal). Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concen-tration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield  were determined from the cell number at the respective observation times.

Three concentrations showed toxicity between 10 and 75 % reduction of yield and be-tween 2 and 30 % inhibition of growth rate compared to the control.

Validation of the analytical method has shown that the substance is not stable under test conditions. Therefore, the content of the test item in the test solutions was determined using GC/FID at the start of the test and every 24 h.

At the beginning of the test, there is a high correlation between nominal and measured concentration: the measured concentrations lay between 87 % and 100 % of the nominal concentrations. But due to insufficient stability of the test item, the measured concentra-tions lay between 51 % and 73 % of the nominal concentrations after 24 h, between 21 % and 40 % of the nominal concentrations after 48 h and between 0 % and 12 % at the end of the test.

In addition, , the scattering values at the end of the test might be caused by the presence of the algae cells (adsorption or ingestion of dissolved test item onto the algae cells). That means a part of the test item was present but not measurable. This effect can be exclud-ed in the highest concentration due to nearly complete inhibition of algae growth.

Therefore, the determination of the results was based on the calculated geometric mean of the measured concentrations in the highest concentration and the respective dilution factor.

The 72h-EC50 values of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were de-termined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

The following results for the test item Naphthalene-1,5-diol were determined:

Endpoint

NOEC

LOEC

EC10

EC50

Growth Rate

1.22 mg/L

(nominal 3.2 mg/L)

2.13 mg/L

(nominal 5.6 mg/L)

1.75 mg/L

> 3.80 mg/L

Yield

1.22 mg/L

(nominal 3.2 mg/L)

2.13 mg/L

(nominal 5.6 mg/L)

1.02 mg/L

2.33 mg/L

Description of key information

One valid experiment was performed.

The following results for the test itemNaphthalene-1,5-diolwere determined:

Endpoint

NOEC

LOEC

EC10

EC50

Growth Rate

1.22 mg/L

(nominal 3.2 mg/L)

2.13 mg/L

(nominal 5.6 mg/L)

1.75 mg/L

> 3.80 mg/L (nominal > 10 mg/L)

Yield

1.22 mg/L

(nominal 3.2 mg/L)

2.13 mg/L

(nominal 5.6 mg/L)

1.02 mg/L

2.33 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
10 mg/L
EC10 or NOEC for freshwater algae:
1.75 mg/L

Additional information