1,4,4-trimethyl-2,3-diazabicyclo[3.2.2]non-2-ene 2,3-dioxide

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:

March 16 to April 20, 2018

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)

Version / remarks:

Adopted March 23, 2006; Annex 5 corrected 28 July 2011

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch 677700
- Expiration date of the lot/batch:13 June 2018
- Purity: 99.8%

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.0, 3.2, 10, 32 and 100 mg/L
- Sampling method: Samples for possible analysis were taken from all test concentrations and the control at t=0, 24, and 72hrs. 1.6 mL of sample was taken from the approximate centre of the test vessels. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling, with the exception of the replicates prepared at the concentration of nominally 1.0 mg/L. For this concentration one of the replicates was sampled individually, while the other two were pooled before sampling.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The highest concentration of the test solution (100 mg/L) was prepared via vigorous shaking to completely dissolve test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
- Controls: Test medium without test item or other additives

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green Algae
- Strain: NIVA CHL 1
- Source: In-house laboratory culture

ACCLIMATION
- Acclimation period: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C. Three to four days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10E4 cells/mL
- Culturing media and conditions (same as test or not): M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). The pre-culture was maintained under the same conditions as used in the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

At the end of the final test microscopic observations were performed on the intermediate concentration of 3.2 mg/L to observe for any abnormal appearance of the algae compared to the control.

Hardness:

24 mg CaCO3/L

Test temperature:

21-24°C

pH:

6.0-9.0

Nominal and measured concentrations:

Nominal concentrations: 1.0, 3.2, 10, 32 and 100 mg/L
Measured concentrations: 0.88, 2.7, 8.5, 26, and 76 mg/L (time-weighted average concentrations)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass
- Type: closed
- Material, size, headspace, fill volume: 50 mL of test solution
- Aeration: No
- Initial cells density: 1 x 10E4 cells/mL
- No. of vessels per concentration (replicates): 3 replicates of each test concentration; 1 extra replicate of each test group for sampling purposes after 24 hours of exposure; 1 or 2 replicates of each test concentration without algae
- No. of vessels per control (replicates): 6 replicates

GROWTH MEDIUM
- Standard medium used: yes; M2 medium prepared according to the OECD 201 Guideline

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 5.4 to 5.9 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL: 1.57 to 1.63 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.93 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.75 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.88 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.88 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the TWA concentration of 2.7 mg/L when compared to the control

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: The 72hr-EC50 for growth rate inhibition was 1.6 mg/L (95% CL: 1.5 to 1.6 mg/L); the historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. The 72hr-EC50 for yield inhibition was 0.50 mg/L (95% CL: 0.48 to 0.52 mg/L); the historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L

Reported statistics and error estimates:

TWA values with 95% confidence intervals

It should be noted, that in Replicate 2 of the nominal 1.0 mg/L concentration group, almost no algae growth was observed throughout the test. The actual concentration measured in this replicate at the end of the test was comparable to the concentrations in the other replicates. Therefore, it can be concluded that the inhibition of growth measured in this replicate was not test item related and thus not relevant. The reason for the lower growth is unknown, a possible explanation can be a mistake during inoculation of the medium. Consequently, this replicate was excluded from data analysis.

 

Study validation results:

- The factor of the biomass parameter, measured in the control between 0 and 72 h, must be at least 16. In the current test it was found to be 184. The test fulfils this validity criterion.

- The coefficient of variation of the mean specific growth rate replicates in the control between 0 and 72 h was: 1.2%. According to OECD 201, the coefficient of variation of the mean specific growth rate, measured in the control from 0 to 72 h, must not exceed 7%. The test fulfils this validity criterion.

- The mean of the replicate coefficients of variation for section-by-section growth rate in the control was: 16%. According to OECD 201, the mean coefficient of variation, measured in the control from 0 to 72 h, must not be higher than 35%. The test fulfils this validity criterion.

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, TAOBN reduced growth rate and inhibited the yield of this fresh water algae species significantly at the Time Weighted Average (TWA) concentration of 0.88 mg/L and higher. The 72-hr EC50 for growth rate inhibition was 5.6 mg/L with a 95% confidence interval ranging from 5.4 to 5.9 mg/L, based on TWA concentrations. The 72hr-EC50 for yield inhibition was 1.60 mg/L with a 95% confidence interval ranging from 1.57 to 1.63 mg/L, based on TWA concentrations.

Description of key information

The EC50 for growth rate inhibition (72h-ErC50) was 5.6 mg/L.

The EC10 for growth rate inhibition (72h-ErC10) was 0.88 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

5.6 mg/L

EC10 or NOEC for freshwater algae:

0.88 mg/L