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:
02 January 2017 through 21 April 2017
Reliability:
1 (reliable without restriction)

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, 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:
Batch No. : VE00466805
Purity : 93.5% (sum of two isomers)
Expiry : 23 January 2019

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
For the determination of the actual test item concentrations, duplicate samples were taken from each test concentration and the control at the start and at the end of the two 24-hour test medium renewal periods.

However, from the highest test concentration of 25mg/L, the last samples were taken after 24 hours, since all daphnids were dead at that observation time at this concentration.

For sampling from the aged test media, the contents of the respective replicates were combined prior to sampling.

All samples were stored frozen (at about -20 °C) immediately after sampling. Based on pre-experiments for investigation of the storage stability (non GLP IES Study 20160321), the test item was found to be stable in the test water under these storage conditions.

The concentrations of GR-50-3010 were analyzed in one of the duplicate test medium samples from the control and all test concentrations with surviving daphnids from the start and end of both test medium renewal periods.

Test solutions

Vehicle:
no
Details on test solutions:
The test medium of the highest nominal concentration of 50 mg/L was prepared by completely
dissolving 50.20 mg of test item in 1000 mL of test water using intense stirring for 24 hours at
room temperature in the dark. Adequate volumes of this test medium were serially diluted with
test water to prepare the lower concentrations of test media.

The test media were prepared just before the start of the test.

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.

An inoculum culture was set up four days before the start of the exposure. The inoculum culture
was diluted threefold one day before the start of the test to ensure that the algae were in the
exponential growth phase when used to inoculate the test solutions.

Study design

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

Test conditions

Hardness:
15 mg/L as CaCO3
Test temperature:
22 °C
pH:
pH 7.3 - 7.8
Nominal and measured concentrations:
The measured concentrations of GR-50-3010 in the test media of the nominal test
concentrations of 0.50 to 50 mg/L were between 90 and 98% of the nominal values at the start
of the test. Thus, the correct dosage of the test item could be verified. During the test period of
72 hours, the test item concentrations in the test media decreased to 42 to 71% of the nominal
values.
Details on test conditions:
75 mL glass Erlenmeyer flasks were used as incubation vessels. The volume of test solution in
each test flask was 30 mL per replicate. Each test flask was covered with a glass dish.

Incubation - The test flasks were incubated in a temperature controlled orbital shaker (Multitron-Pro, Infors
HT, Bottmingen/Switzerland) at a temperature of 22 °C. The test flasks were positioned
randomly and repositioned daily.

Light Intensity - They were continuously illuminated by LED light installed
above the test flasks. The light intensity at the level of the test solutions was approximately
64 μE s-1 m-2 (range: 63 to 66 μE s-1 m-2, measured at nine places in the experimental area). The
light intensity over the incubation area was within a ±15%-deviation from the average light
intensity as recommended by the guideline.

Based on the results of the range-finding experiment and in agreement with the Sponsor the following nominal concentrations
of GR-50-3010 were selected for the main test: 0.50, 1.6, 5.0, 16 and 50 mg/L. Additionally, a
control (test water without test item) was tested in parallel.

The test design included three replicates per test concentration and six replicates of 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).
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
10.8 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI : 9.9 - 11 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
5 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI : 3.6 - 6.1 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
6.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other:
Remarks:
95% CI : 5.4 - 7.1 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.5 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 95% CI : 1.9 - 3.0 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.02 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.02 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Results with reference substance (positive control):
The 72-hour EC50 for growth rate in the reference test IES Study Number 20160277 was 0.9 mg/L (October 2016)
and showed that the sensitivity of the test system was within the range recommended by the
guideline (72-hour EC50 for the growth rate 0.9-1.5 mg/L).

Any other information on results incl. tables

A clear concentration-response relationship was observed for both biological parameters

growth rate and yield during the exposure period.

A statistically significant inhibitory effect on the growth rate and yield of the algae was

observed at the end of the test at the mean measured concentration of 3.9 mg/L and at all higher

test concentrations (results of Welch t-test, one-sided smaller, α = 0.05).

Therefore, the NOEC for growth rate and yield was determined to be at the test concentration

of 1.02 mg/L. The LOEC for growth rate and yield was determined to be 3.9 mg/L.

The recoveries observed at the end of the test in the additional samples incubated without algae

and in the dark were in the same range and both higher than in the samples containing algae,

showing a potential adsorption of the test item onto the algae (23%) but no photolytic

degradation of the test item.

The microscopic examination of the algal cells at the end of the test showed no difference

between the algae growing at the nominal test concentration of 1.6 mg/L (mean measured

1.02 mg/L) determined as the NOEC, and the algal cells in the control. The shape and size of

the algal cells were obviously not affected by the test item up to at least the NOEC.

No remarkable observations were made concerning the appearance of the test media. All test

media were clear solutions throughout the test period.

All validity criteria were fulfilled :

In the control, the biomass increased by a factor of 112 over 72 hours. The validity criterion of

increase of biomass by at least a factor of 16 within three days was fulfilled.

The mean coefficient of variation of the daily growth rates in the control (section-by-section

growth rates) during 72 hours was 15%. According to the OECD test guideline, the mean

coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled.

The coefficient of variation of the average specific growth rates in the replicates of the control

after 72 hours was 1.1%. According to the OECD test guideline, the coefficient of variation

must not be higher than 7%. Thus, the validity criterion was fulfilled

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
End-points determined for GR-50-3010 in a full-GLP OECD 201 Algal Inhibition study, based on mean measured time-weighted average exposure concentrations :
Based on Growth Rate -
72hr ErC50 = 10.8 mg/L
72hr ErC10 = 5.0 mg/L
72hr NOErC = 1.02 mg/L

Based on Yield -
72hr EyC50 = 6.1 mg/L
72hr EyC10 = 2.5 mg/L
72hr NOEyC = 1.02 mg/L
Executive summary:

End-points determined for GR-50-3010 in a full-GLP OECD 201 Algal Inhibition study, based on mean measured time-weighted average exposure concentrations :

Based on Growth Rate -

72hr ErC50 = 10.8 mg/L

72hr ErC10 = 5.0 mg/L

72hr NOErC = 1.02 mg/L

Based on Yield -

72hr EyC50 = 6.1 mg/L

72hr EyC10 = 2.5 mg/L

72hr NOEyC = 1.02 mg/L