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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:
16 - 19 Mar 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Physical state: viscous colorless liquid at room temperature (22-23 °C)
- Source and lot/batch No.of test material: Lot 10115
- Expiration date of the lot/batch: 10 Jan 2019
- Purity: 95.6%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: frozen in darkness

FORM AS APPLIED IN THE TEST: solid. The test material did not liquefy after being brought to room
temperature.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: All; sampled at test initiation (0 hours) and at test termination (72 hours).
- Sampling method: Samples were processed immediately for analysis. Samples collected at test initiation (0 hour) were collected from the individual batches of test solution prepared for each treatment and control group prior to distribution into the test chambers. At exposure termination (72 hours), samples were collected from the pooled replicates from each treatment and control group and the single abiotic replicate in the highest treatment group. At each sampling interval, 8.0 mL of test solution was added to 20 mL glass scintillation vials containing 2.0 mL of 0.5% formic acid in methanol.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A primary stock solution was prepared by mixing a calculated amount of test substance in 4 L of dilution water at a nominal concentration of 100 mg formulation/L, the highest concentration tested. The stock solution concentration was not adjusted for the active ingredient of the test substance during preparation, and all concentrations are based on the test substance as received. The test substance was weighed onto a Teflon® disk, added to a 4 L WAF bottle containing 4 L of dilution water, and stirred for approximately four hours on a magnetic stir plate. A piece of neoprene was placed between the bottle and the stir plate to minimize the potential of a temperature change in the stock solution. The stock solution appeared colorless with a white precipitate in the water column. The stock solution was allowed to settle briefly to allow the precipitate to settle out. The solution was drawn off through a spigot and tubing placed approximately 2-3 cm from the bottom of the bottle (mid-depth). Aliquots of the primary stock solution were proportionally diluted with dilution water to a final volume of 0.5 L to prepare four additional test solutions at nominal concentrations of 6.3, 13, 25 and 50 mg formulation/L. The solutions were inverted to mix and appeared clear and colorless and otherwise unremarkable at the time of preparation.
- Controls: freshwater AAP medium without test substance
- Evidence of undissolved material: No

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: not reported
- Source (laboratory, culture collection): Original algal cultures were obtained from the University of Texas at Austin, and have been maintained in culture medium at EAG Laboratories, Easton, Maryland since June 2017.
- Age of inoculum (at test initiation): Algal cells were inoculated four days before the test from an actively growing culture medium which had been maintained under similar environmental conditions as used in this test for at least two weeks prior to test initiation
- Method of cultivation: Stock nutrient solutions were prepared by adding reagent-grade chemicals to purified EAG Laboratories-Easton well water (NANOpure® water). The test medium then was prepared by adding appropriate volumes of the stock nutrient solutions to NANOpure® water. The pH of the medium was adjusted to 7.5 ± 0.1 with 10% hydrochloric acid and/or 0.1N sodium hydroxide, as needed, at the time of preparation. The medium was sterilized by filtration (0.22 μm) and stored refrigerated prior to use.

Study design

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

Test conditions

Test temperature:
23.81 - 24.12 °C
pH:
7.3 - 9.4
Nominal and measured concentrations:
Nominal: 6.3, 13, 25, 50 and 100 mg formulation/L
Measured: 0.017, 0.37, 1.2, 2.7 and 5.1 mg/L (geometric mean)
Details on test conditions:
TEST SYSTEM
- Test vessel: sterile 250-mL glass Erlenmeyer flasks plugged with sterile foam stoppers
- Type: closed
- Fill volume: 100 mL
- Aeration: continuous shaking at 100 RPM
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 315E+04 cells/mL
- No. of vessels per concentration (replicates): three
- No. of vessels per control (replicates): six

GROWTH MEDIUM
- Standard medium used: yes, AAP medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: purified well water (NANOpure®)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 5,550 to 6,120 lux (measured at test solution level at five locations surrounding the test flasks at test initiation using a SPER Scientific 840006C light meter)

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: electronic particle counter (Z2; Coulter Electronics, Inc.), appearance of cells
- Recovery phase: At the end of the 72-hour exposure period, those treatment levels that exhibited approximately 50% or greater reduction in mean cell density relative to the blank control were selected for a recovery test. One recovery replicate per selected treatment level and negative control were maintained under environmental conditions similar to those in the exposure period. In order to initiate the recovery test, a 0.5 mL aliquot from each of the three replicates in the selected treatment levels were transferred to a clean flask with fresh medium. For the negative control group, a 0.5 mL aliquot was transferred from one indiscriminately selected blank control replicate to a clean flask with fresh medium. Samples of the recovery replicates were collected on days 0 and 4 of the recovery phase and were counted at the end of the recovery phase.

RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0, 10, and 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 5.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: Test conducted at a loading rate of 100 mg/L.
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: none. After 72 hours of exposure, aggregation, flocculation, or adherence to the test chambers was not observed in the controls or in any treatment groups. There were no noticeable changes in cell morphology in any of the treatment groups when compared to the control replicates during the microscopic examinations of the cells.
- Recovery phase: To determine if MTDID 10628 had an algistatic or algicidal effect, a recovery test was initiated for the 2.7 and 5.1 mg formulation/L after the 72-hour exposure. In the recovery phase it was demonstrated that the effects of MTDID 10628 were algistatic (reversible) rather than algicidal, to R. subcapitata at concentrations less than or equal to 5.1 mg formulation based on greater than 16X increase of healthy cells within 72 hours.

Any other information on results incl. tables

Table 1. Mean Growth Rate and Percent Inhibition

Geometric Mean, Measured Concentration (mg formulation/L)

0-24 hours
Mean Growth Rate (per hour)

0-24 hours
Percent Inhibition

0-48 hours
Mean Growth Rate (per hour)

0-48 hours
Percent Inhibition

0-72 hours
Mean Growth Rate (per hour)

0-72 hours
Percent Inhibition

Negative Control

0.0832

--

0.0815

--

0.0798

--

0.017

0.0801

4

0.0809

1

0.0792

1

0.37

0.0782

6

0.0811

1

0.0792

1

1.2

0.0744

11

0.0778

5

0.0760*

5

2.7

0.0660

21

0.0720

12

0.0689*

14

5.1

0.0451

46

0.0567

30

0.0562*

30

*Treatment group mean was significantly reduced (Dunnett’s test, p < 0.05) when compared to the negative control mean. Statistical significance only evaluated at 72 hours of exposure.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Control cell density increased by a factor of > 16 (315); the mean CV for section-by-section specific growth rate in the control was < 35% (5.52%); the CV of average specific growth rates in the whole test period for control was < 7% (1.63%).
Conclusions:
The 72-hour EC50 (growth rate) of 1,1'-Isophthaloylbis(2-methylaziridine) to Pseudokirchneriella subcapitata was > 100 mg/L (nominal concentration) (OECD 201). This corresponds to a geometric mean measured concentration of 5.1 mg/L.
Executive summary:

The 72-hour EC50 (growth rate) of 1,1'-isophthaloylbis(2-methylaziridine) to Pseudokirchneriella subcapitata was examined in a static test conducted according to OECD 201. Test solutions were prepared as the control (0 mg/L) and at 6.3 mg/L, 13 mg/L, 25 mg/L, 50 mg/L, and 100 mg/L corresponding to geometric mean measured concentrations of 0.017 mg/L, 0.37 mg/L, 1.2 mg/L, 2.7 mg/L, and 5.1 mg/L. The 50% effect level was not reached at the highest concentration tested. After 72 hours, the ErC50 of 1,1'-isophthaloylbis(2-methylaziridine) was reported as > 5.1 mg/L (geometric mean measured concentration) and the EC10 (growth rate) was reported as 2.2 mg/L (geometric mean measured concentration). The study was well-documented, followed an international standard method, and was GLP compliant; therefore, the test is considered reliable without restriction.