1,3-Propanediaminium, 2-hydroxy-N1,N1,N1,N3,N3-pentamethyl-N3-[3-[(2-methyl-1-oxo-2-propen-1-yl)amino]propyl]-, chloride (1:2)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 30 October 2015 To 09 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: CY50561001
- Expiration date of the lot/batch: 27 June 2017
- Purity: 65.4% (for the substance in solution)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, below 70 RH%)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All
- Sampling method: In all cases (0h, 24h, 48h, 72h), determinations of Test Item concentrations were performed from all replicate vessels at each test concentration and the control. At each sampling occasion, six samples were taken from each concentration and from the control into plastic tubes (approximately 2 mL/sample).
- Sample storage conditions before analysis: Samples were stored at approximately -20°C until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution with a concentration of 100 mg a.i./L (mg active ingredient/L) (nominal) was prepared with Test Item and algal growth medium (OECD Medium).
The test solutions were prepared by appropriate diluting of this stock solution and these solutions were distributed into the appropriate test flasks.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648;
ATCC 22662)
- Source (laboratory, culture collection): The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd.
- Age of inoculum (at test initiation):
- Method of cultivation: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines.

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Culture temperature was checked at the beginning of the experiment and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was in the range of 22.6 – 22.9 °C measured in the flask and between 22.3 and 23.4 °C measured within the climate chamber.

pH:

The pH was checked at the beginning and at the end of the test, in the control and each concentration. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.28 – 8.50 during the experiment.

Nominal and measured concentrations:

Nominal concentrations: 6.25, 12.5, 25.0, 50.0 and 100 mg a.i./L
Measured concentrations: The measured concentration not deviated more than 20% of the nominal.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (250 mL)
- Initial cells density: 10 000 algal cells per mL test medium
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes


OTHER TEST CONDITIONS
- Light intensity and quality: 8418 lux (equivalent to ~114 μE/m2/s)


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.1, 1, 10 and 100 mg a.i./L
- Results used to determine the conditions for the definitive study:
Untreated control: 76.33 (average of cell number at 72 hours (x 10 000 cell/ml))
0.1 mg a.i./L: 72.00 (average of cell number at 72 hours (x 10 000 cell/ml))
1 mg a.i./L: 71.00 (average of cell number at 72 hours (x 10 000 cell/ml))
10 mg a.i./L: 69.00 (average of cell number at 72 hours (x 10 000 cell/ml))
100 mg a.i./L: 35.00 (average of cell number at 72 hours (x 10 000 cell/ml))

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

62 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95 % confidence limits: 53.11 - 72.38 mg a.i./L)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

57.94 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: yield

Remarks on result:

other: (95 % confidence limits: 51.34 - 65.40 mg a.i./L)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Growth rate/ Biomass/ Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Growth rate/ Biomass/Yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): There were no morphological deviations of the algal cells during the study in any of the test groups.
- Other: The 72h EC50 (growth rate) could not be calculated due to the low percent of inhibition.

Results with reference substance (positive control):

The date of the last study (Study Code: 16/281-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 13 – 16 September 2016.
The 72h ErC 50: 0.89 mg/L, (95 % confidence limits: 0.82 – 0.97 mg/L)
The 72h EbC 50: 0.53 mg/L, (95 % confidence limits: 0.49 – 0.58 mg/L)
The 72h EyC 50: 0.62 mg/L, (95 % confidence limits: 0.57 – 0.67 mg/L)
These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).

Reported statistics and error estimates:

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (α = 0.05) by TOXSTAT software.
For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by 2 Sample t-Test (α = 0.05) by TOXSTAT software.

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of this study the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group in the examined concentration range of 50–100 mg a.i./L (nominal), therefore the overall LOEC was determined as 50 mg a.i./L (nominal) and the NOEC was determined as 25 mg a.i./L (nominal).

Executive summary:

The effect of Diquat Test Item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours.

Because toxic response was observed during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.0) and one control were tested in the main experiment. The concentrations in the definitive test were based on the results of the preliminary range-finding test and were: 6.25, 12.5, 25.0, 50.0 and 100 mg a.i./L.

The measured concentration not deviated more than 20% of the nominal therefore, the biological results are based on the nominal test item concentrations.

The test design included three replicates at each test concentration and six replicates for the untreated controls.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software.

The EbC50 and EyC50 values of the Test Item and their confidence limits were calculated using Probit analysis by TOXSTAT software.

In conclusion, under the conditions of this study the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group in the examined concentration range of 50–100 mg a.i./L (nominal), therefore the overall LOEC was determined as 50 mg a.i./L (nominal) and the NOEC was determined as 25 mg a.i./L (nominal).

Description of key information

Exponentially growing cultures of Pseudokirchneriella subcapitata were exposed to various concentrations of the Test Item over several generations under defined conditions (according to OECD TG 201 and EU Method C.3).

No deviation from the guideline was observed during the test.

Key value for chemical safety assessment

EC50 for freshwater algae:

57.94 mg/L

EC10 or NOEC for freshwater algae:

25 mg/L

Additional information

The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h average specific growth rate was statistically significantly different from the untreated control value in the concentration range of 50-100 mg a.i./L. Therefore, the LOEC was considered to be 50 mg a.i./L, correspondingly the NOEC was determined as 25 mg a.i./L. The 72 h EC50 could not be calculated due to the low percent of inhibion.