Registration Dossier

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:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Lot No. : 43
Purity : 96.5%
Expiration date : September 05, 2017

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
For the determination of the actual test item concentrations, duplicate samples were taken from
each treatment at the start of the test.
After 24, 48 hours and at the end of the test (after 72 hours), stability samples (containing algae)
were taken in duplicate from all test concentrations and from the control.
The stability samples at 24 and 48 hours (duplicates 50 mL samples) could not be taken from
the test vessels itself, as the principle of a closed system is, that the test vessels have to remain
completely filled with test medium during the entire test period. Therefore, for this sampling at
24 and 48 hours, a set of additional flasks containing the corresponding test medium (with
algae) was incubated under conditions identical to the test.
For sampling at the end of the test, the test medium of the treatment replicates was pooled.
Additional test vessels with test medium at the dilution 1:2.3 were incubated under the
following conditions:
- Test medium without algae under light conditions in order to document possible
adsorption of the test item onto algae.
- Test medium without algae in the dark in order to document a possible photolytic
degradation of the test item.
All samples were stored frozen (at -20 ± 5 °C) immediately after sampling. Based on preexperiments
for investigation of the storage stability, the test item was found to be stable in the
test water under these storage conditions.
The concentrations of GR-86-6599 were analytically measured in one of the duplicate samples
taken from all sampling dates. From the additional flasks without algae, one of the duplicate
samples were measured after 24 and 72 hours.

Test solutions

Vehicle:
no

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test organism used for the study was Pseudokirchneriella subcapitata (formerly
Selenastrum capricornutum, occasionally also listed as Raphidocelis subcapitata),
Strain No. 61.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant
Physiology, University of Göttingen, 37073 Göttingen / Germany). The algae were cultivated
at IES Ltd Laboratories under standardized conditions according to the test guidelines.

Study design

Test type:
static
Water media type:
freshwater
Remarks:
Reconstituted test water (AAP Medium)
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
15 mg/L
Test temperature:
22°C
pH:
7.6 - 7.9
Nominal and measured concentrations:
Based on these results and in agreement with the Sponsor the following nominal concentrations
of GR-86-6599 were selected for the main test: the saturated test medium with a loading rate of
50 mg/l and dilutions 1:5.1, 1:3.4, 1:2.3 and 1:1.5. Additionally, a control (test water without
test item) was tested in parallel.
Details on test conditions:
The main test was performed in a closed system. The test design included three replicates per
test concentration and six replicates for the control.
The test was started using a nominal algal cell density of 5000 cells/mL. The initial cell density
was selected according to the recommendations of the OECD test guideline. The algal cell
density in the pre-culture was determined using an electronic particle counter (Coulter
Counter, Model Z2).
A static test design was applied. The duration of the test was 72 hours.
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 22 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
This exposure concentration represents the maximum water saturation concentration of the test item
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 22 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
This exposure concentration represents the maximum water saturation concentration of the test item
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 22 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
This exposure concentration represents the maximum water saturation concentration of the test item
Details on results:
No effects observed on algal growth rate up to and including the highest tested exposure concentration, equivalent to the saturation concentration.
Results with reference substance (positive control):
Potassium Dichromate : 72 hour EC50 (growth rate) = 0.9 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Endpoint after 72 Hours Growth Rate [μg/L] Yield [μg/L]
EC10 >22.0 16.6
(95% Confidence Interval) n.d. (15.7-17.5)
EC20 >22.0 >22.0
EC50 >22.0 >22.0
NOEC 22.0 16.1
LOEC n.d 16.7
n.d.: Not determined.
Executive summary:

The impact of the test item GR-86-6599 on the growth of the freshwater green algal species

Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the

OECD Guideline 201 (2006, corrected 2011) and the Commission Regulation (EU)

No 2016/266, C.3.

As the test item is a volatile substance, the test was performed using glass stoppered Erlenmeyer

flasks (closed system) completely filled with test medium, minimizing the air space in the flasks

and avoiding potential losses of test item by evaporation.

As the test item is a liquid with low water solubility, the slow stirring method was applied for

preparation of a saturated test item solution. For preparation of the highest concentrated test

medium, the test item was carefully applied (pipetted) onto the surface of the test water at a

loading rate of 50 mg/L. Thereafter slow stirring was applied for 48 hours in a closed vessel to

reach a maximum concentration of dissolved test item in the test water. The mixing vessel was

nearly completely filled (a small headspace had to be included as the test item was floating on

the water surface) and tightly sealed with glass stoppers. After this treatment, stirring was

stopped for 24 hours as a precaution to allow phase separation in case test item droplets had

been mixed into the water column during stirring. Following cessation of mixing and the period

of settling, the lower aqueous and equilibrated phase was carefully separated from the nondissolved

upper test item phase through the tap at the bottom of the vessel. This equilibrated

aqueous phase with a loading rate of 50 mg/L, containing dissolved test item only, was used as

the highest test concentration and considered to represent the aqueous saturation concentration

in test media of the test item. The highest test concentration was subsequently diluted with test

water to obtain the following test media: the dilutions 1:5.1, 1:3.4, 1:2.3 and 1:1.5. Additionally,

a control (test water without test item) was tested in parallel.

The preparation of the test media was based on the OECD Guidance Document No. 23 on

Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

No significant algacidal effects of the test item were observed on the growth rate component at

test concentrations up to and including the highest exposure concentration which corresponded

to the saturation concentration in test media of the test item. Only minor statistically significant

effects on the yield of algae were observed in this study with only 18% inhibition occurring at

the saturation exposure concentration