Methyl 2,3-dibromopropionate

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 2019-02-11 to 2019-10-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Batch No.: 800327680
Purity: 98%

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L, and control
- Sampling method: 2.0 mL from the approximate centre of the test vessels at the start of the test
- Sample storage conditions before analysis: Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with the highest concentration of 100 mg/L in the first range-finder and 10 mg/L in the second range-finder and final test, applying an overnight period of magnetic stirring to accelerate dissolution of test item in test medium. In the first range-finder, a few particles of undissolved material were observed at the surface of the solution. Since the test item is a liquid, these solid particles are probably related to salts in the test medium. No such particles were observed at the highest concentration prepared (i.e. 10 mg/L) in the second range-finder and final test. Therefore, the test item was considered to be completely dissolved and lower test concentrations were prepared by subsequent dilution of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Method of cultivation: 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.
- Light intensity: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
- Pre-culture: Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of
1E+4 cells/mL. 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

Hardness:

24 mg CaCO3/L

Test temperature:

22-23 °C

pH:

7.9-8.3

Nominal and measured concentrations:

Nominal: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution.
- Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
- Initial cells density: 1E+04 cells/m
- Control end cells density: 3.433 E+06 cells/mL
- Replicates: 3 replicates of each test concentration, 6 replicates of the control, 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.


GROWTH MEDIUM
- Standard medium used: yes, standard M2


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition: NH4Cl: 15 mg/L; MgCl2.6H2O: 12 mg/L; CaCl2.2H2O: 18 mg/L; MgSO4.7H2O: 15 mg/L; KH2PO4: 1.6 mg/L; FeCl3.6H2O: 64 μg/L; Na2EDTA.2H2O: 100 μg/L; H3BO3: 185 μg/L; MnCl2.4H2O: 415 μg/L; ZnCl2: 3 μg/L; CoCl2.6H2O: 1.5 μg/L; CuCl2.2H2O: 0.01 μg/L; Na2MoO4.2H2O: 7 μg/L; NaHCO3: 50 mg/L
- Culture medium different from test medium: no


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Continuously using TLD-lamps
- Light intensity and quality:86 to 90 μE.m-2.s-1.
- Salinity (for marine algae):

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement:
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- First Range-Finding Test: Three replicates of exponentially growing algal cultures were exposed to a range of 0.10 to 100 mg/L increasing by a factor of 10 and to a blank control.
Concentration-related inhibition of algal growth rate and yield was found at all test concentrations, resulting in 100% growth rate and yield inhibition at 10 and 100 mg/L. At the lowest concentration tested, 20% growth rate inhibition and 48% yield inhibition was recorded.
- Second Range-Finding Test: Three replicates of exponentially growing algal cultures were exposed to a range of 0.0010 to 1.0 mg/L increasing by a factor of 10 and to a blank control.
Stimulation of algal growth was found at the two lowest test concentrations, while growth rate was inhibited by 3.8 and 79% at concentrations of 0.10 and 1.0 mg/L, respectively. Yield was reduced by 19 and 99% at these concentrations. Based on the obtained results, the 72h-EC50 for both growth rate and yield is expected to lay between 0.10 and 1.0 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.74 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Measured Test Item Concentrations:
No test item response was measured in any of these samples, and consequently, no further analyses were considered. It was questioned what caused this artefact considering that the limit of detection was 0.00023 mg/L on the day of analysis. One possible cause was investigated, namely that the test item had completely degraded in test medium prior to the start of the exposure phase. An additional analytical trial was performed for this purpose.
Although exposure concentrations are not analytically confirmed, the test item was completely soluble in test medium upon preparation of test solutions.

- Analytical Trial:
The measured concentrations at the start of the analytical trial were 0.046 and 0.82 mg/L (being the means of duplicate analysis) in QC samples prepared at 0.10 and 1.0 mg/L, respectively. The concentrations were approximately 15 and 20 times lower after 3 hours of incubation relative to the initial values, respectively. These concentrations were below the lowest calibration point, and therefore, could not be quantified reliably. All test item responses detected at both QC concentrations were at carry-over level after 6, 16 and 24 hours of incubation. Therefore, it was assumed that the test item had completely degraded during the first 6 hours.
Based on the obtained results, no exact half-life times could be calculated as no test item concentrations were quantified in any of the samples analysed after test commencement. However, when assuming that the concentrations analysed after three hours of incubation were 15 and 20 times lower than the initial values (i.e. 0.0031 and 0.041 mg/L, respectively), the DT50 was determined to be shorter than 1 hour at both QC levels.

- Inhibition of Growth Rate and Inhibition of Yield:
Inhibition of algal growth increased with increasing concentration from 0.032 mg/L upwards, resulting in 61% growth rate inhibition and 95% yield reduction at the highest concentration tested. Statistically significant inhibition of growth rate inhibition was considered to be biologically not relevant at 0.032 mg/L, where the observed inhibition was below 10%. The NOEC based on biological relevance was thus 0.032 mg/L.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 0.32 mg/L when compared to the control.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: The 72h-EC50 for growth rate inhibition (ERC50) was 1.97 mg/L with a 95% confidence interval ranging from 1.93 to 2.00 mg/L.

Reported statistics and error estimates:

For determination of the NOEC and the ECx-values the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate (Step-down Jonckheere-Terpstra Test, α=0.05, one-sided, smaller) or inhibition of yield (Williams Multiple Sequential t-test, α=0.05, one-sided, smaller).
Calculation of ECx-values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding concentrations of the test item.
ToxRat Professional v 3.2.1 (ToxRat Solutions GmbH, Germany) was used to perform the analysis.

Effect Parameters:

Parameter(mg/L)		NOEC*	NOEC	EC10	EC20	EC50
Growth rate	Value	0.010	0.032	0.16	0.27	0.74
	lower 95%-cl			0.066	0.15	0.58
	upper 95%-cl			0.25	0.37	1.0
Yield	Value	0.010	0.010	0.027	0.049	0.15
	lower 95%-cl			0.020	0.040	0.13
	upper 95%-cl			0.034	0.058	0.17

cl – confidence limit, * - based on statistical significance, # - based on biological relevance.

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata, test item inhibited growth rate and yield of this freshwater algae species significantly at nominal concentrations of 0.032 mg/L and higher.
The 72h-EC50 for growth rate inhibition (ERC50) was 0.74 mg/L with a 95% confidence interval ranging from 0.58 to 1.0 mg/L. The 72h-EC50 for yield inhibition (EYC50) was 0.15 mg/L with a 95% confidence interval ranging from 0.13 to 0.17 mg/L. The 72h-NOEC for growth rate inhibition was 0.010 mg/L based on statistical significance and 0.032 mg/L based on biological relevance. The 72h-NOEC for yield inhibition was 0.010 mg/L based on both statistical significance and biological relevance.
It should be noted that the test item degraded completely prior to the start of the exposure and that concentrations of possible degradation products were not quantified during this project due to the absence of an appropriate analytical method.

Executive summary:

The objective of the study was to evaluate test item for its ability to generate toxic effects in Raphidocelis subcapitata during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10, EC20 and EC50 for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on OECD 201 and OECD 23.

Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to nominal concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L. The initial algal cell density was 1E+04 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start of the test.

 

Under the conditions of the present study with Raphidocelis subcapitata, test item inhibited growth rate and yield of this freshwater algae species significantly at nominal concentrations of 0.032 mg/L and higher.

The 72h-EC50 for growth rate inhibition (ERC50) was 0.74 mg/L with a 95% confidence interval ranging from 0.58 to 1.0 mg/L. The 72h-EC50 for yield inhibition (EYC50) was 0.15 mg/L with a 95% confidence interval ranging from 0.13 to 0.17 mg/L. The 72h-NOEC for growth rate inhibition was 0.010 mg/L based on statistical significance and 0.032 mg/L based on biological relevance. The 72h-NOEC for yield inhibition was 0.010 mg/L based on both statistical significance and biological relevance.

It should be noted that the test item degraded completely prior to the start of the exposure and that concentrations of possible degradation products were not quantified during this project due to the absence of an appropriate analytical method.

Description of key information

The toxic effects in Raphidocelis subcapitata during an exposure period of 72 hours were assesssed based on OECD 201 and OECD 23.

Under the conditions of the present study with Raphidocelis subcapitata, test item inhibited growth rate and yield of this freshwater algae species significantly at nominal concentrations of 0.032 mg/L and higher.

The 72h-EC50 for growth rate inhibition (ERC50) was 0.74 mg/L with a 95% confidence interval ranging from 0.58 to 1.0 mg/L. The 72h-EC50 for yield inhibition (EYC50) was 0.15 mg/L with a 95% confidence interval ranging from 0.13 to 0.17 mg/L. The 72h-NOEC for growth rate inhibition was 0.010 mg/L based on statistical significance and 0.032 mg/L based on biological relevance. The 72h-NOEC for yield inhibition was 0.010 mg/L based on both statistical significance and biological relevance.

It should be noted that the test item degraded completely prior to the start of the exposure and that concentrations of possible degradation products were not quantified during this project due to the absence of an appropriate analytical method.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.74 mg/L

EC10 or NOEC for freshwater algae:

0.01 mg/L