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:
30 October 2017- 21 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 201

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Analytical grade is identical to the technical grade
Specific details on test material used for the study:
Batch No.: 10013913. Active ingredient purity: >99.9 % (analytical). White to light beige powder. Expiry date: 30 November 2018. Storage conditions at test facility: 4± 4 °C, in the dark.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The samples were taken from the biological phase of the study. Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at the start of the test. For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment. The samples were diluted by a factor of two with acetonitrile. One additional sample of the control was taken at test start and test end without any sample treatment.

Test solutions

Vehicle:
no
Details on test solutions:
A stock solution of 20 mg test item/L was prepared by dissolving 20.2 mg test item into 1010 mL test water by intense stirring for 60 minutes and ultrasonic treatment for one minute. The pH value of the stock solution was adjusted with 1 M NaOH to 8.1. Adequate volumes of this stock solution were diluted with test water to prepare the test media of the desired test concentrations of 1.00, 0.32, 0.10, 0.032 and 0.01 mg test item/L. Additionally, a control consisting of reconstituted water (OECD medium) was also set up.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV). The algae were supplied by the "Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany. The algae were cultivated at the test facility under standardised conditions according to the test guidelines.

Study design

Test type:
static
Water media type:
other: Reconstituted water
Remarks:
OECD Medium
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
Water temperature ranged from 21.0 to 21.5 °C
pH:
8.0 to 8.1 at test start and 8.0 to 9.2 at test end.
Nominal and measured concentrations:
1.00, 0.32, 0.10, 0.032 and 0.01 mg test item/L and a control, corresponding to the following geometric mean measured concentrations of the test item: 1.00, 0.28, 0.047, 0.012 and 0.004 mg test item/L, and a control.
Details on test conditions:
Light regime: Continuous illumination
Mean light intensity: 5690 Lux (range: 4900 to 6200 Lux)
Reference substance (positive control):
yes
Remarks:
The reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.289 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: <0.004-n.d
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.047 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The 72-hour EyC50 was calculated to be 0.112 mg test item/L and the ErC50 0.289 mg test item/L. The 72-hour EyC10 was calculated to be 0.044 mg test item/L and the ErC10 0.208 mg test item/L. The 72-hour NOEyC was determined to be 0.047 mg test item/L and the associated 72-hour LOEyC of 0.28 mg test item/L. The 72-hour NOErC was determined to be 0.047 mg test item/L and the associated 72-hour LOErC was 0.28 mg test item/L.
At the start of the test 98% of the nominal test concentrations were found (average of all test concentrations). After 72 hours test duration, 45% of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 72% of nominal.
Results with reference substance (positive control):
The most recent test with the reference item potassium dichromate was performed in July 2017. As a result, the 72-h EC50 values were determined to be 0.415, 1.02 and 0.455 mg test item/L based on yield, growth rate and biomass, respectively.

Any other information on results incl. tables

The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal test concentration of 1.0 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The influence of 2-Methyl-4-Isothiazolin-3-One Hydrochloride on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a 72 -hour static concentration-response test. The 72-hour EyC50 was calculated to be 0.112 mg test item/L and the 72-hour ErC50 value was calculated to be 0.289 mg test item/L. The 72-hour NOEyC was determined to be 0.047 mg test item/L and the associated 72-hour LOEyC was 0.28 mg test item/L. The 72-hour NOErC was determined to be 0.047 mg test item/L and the associated 72-hour LOErC was 0.28 mg test item/L. All reported results refer to geometric mean measured concentrations, since the test item concentrations were not within ± 20% of the nominal concentrations during the test.
Executive summary:

The purpose of this test was to determine the inhibitory effect of the test item 2-Methyl-4-Isothiazolin-3-One Hydrochloride on the growth of the freshwater green algae Pseudokirchneriella subcapitata. For this purpose, exponentially growing cultures of this unicellular green algal species were exposed for 72 hours to various concentrations of the test item under defined conditions. The test concentrations were 1.00, 0.32, 0.10, 0.032 and 0.01 mg test item/L, and a control (0.0 mg test item/L) consisting of OECD medium. The test was started (0 hours) by inoculation of a biomass of approx. 5000 algal cells per mL test medium and consisted of three replicates per test concentration and six replicates in the control. Volumes of 50 mL of algal suspension per replicate were continuously stirred with magnetic stirrers in 50 mL Erlenmeyer flasks. The cell density on each observation time was determined by spectrophotometric measurement after 24, 48 and 72 hours of exposure. Based on the calculated cell densities, the 72-hour ErC50 and the 72-hour EyC50, the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by a 3-parametric concentration distribution function. The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

All validity criteria were met. The 72-hour EyC50 was calculated to be 0.112 mg test item/L and the 72-hour ErC50 value was calculated to be 0.289 mg test item/L. The 72-hour NOEyC was determined to be 0.047 mg test item/L and the associated 72-hour LOEyC was 0.28 mg test item/L. The 72-hour NOErC was determined to be 0.047 mg test item/L and the associated 72-hour LOErC was 0.28 mg test item/L. All reported results refer to geometric mean measured concentrations, since the test item concentrations were not within ± 20% of the nominal concentrations during the test.