Bromoform

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:

Experimental Phase:12 January 2018 to 23 July 2018. Report Issued: 07 November 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for immediate quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 24 ± 1 °C.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 ±1 °C

pH:

The pH value of the control cultures was observed to increase from pH 7.9 at 0 hours to pH 10.2 at 72 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the transfer rate of CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. The increase in pH after 72 hours was in excess of that recommended in the Test Guidelines (1.5 pH units after 72 hours). This was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion given in the Test Guidelines.

Nominal and measured concentrations:

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.26 to 34 mg/L (26% to 55% of the nominal test concentrations). Analysis of the test preparations at 72 hours showed measured test concentrations to range from 0.25 to 36 mg/L (91% to 108% of the 0-Hour measured test concentrations) and as such it was considered appropriate to calculate the results based on the 0-Hour measured test concentrations.

It was considered likely that the failure to obtain near nominal concentrations in the 0-Hour test preparations was due to losses through volatilisation during the time taken to prepare and sample the test solutions. Failure to achieve near nominal concentrations was considered to have had no impact on the outcome of the test given that a concentration dose response was observed allowing the calculation of both EC50 and NOEC values at the concentrations achieved.

Details on test conditions:

TEST SYSTEM (DEFINITIVE TEST)
- Test vessel: 250mL glasss conical flasks
- Type (delete if not applicable): closed - flasks were sealed with ground glass stoppers
- Fill volume: completely filled
- Aeration: Oribital shaker (100 - 150 rpm)
- Renewal rate of test solution (frequency/flow rate): none (Static test)
- Initial cells density: 5,000 cells/mL
- Control end cells density: 625,00 cells/mL at 72 hours
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes - The culture medium was prepared using reverse osmosis purified deionised water and the pH adjusted to 7.5 with 0.1N NaOH or HCl. For the purposes of the range-finding test, initial experiments and definitive test, additional sodium bicarbonate (250 mg/L) was added to the prepared culture medium prior to use.


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: Yes, for culture medium
- Photoperiod: Cultures were continuously illuminated
- Light intensity and quality: warm whirte light (380 - 730 nm) at approximately 7000 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
- Determination of cell concentrations: Electronic particle counter

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.1, 1, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: Yes

- Definitive Test
- Test concentrations:1.0, 3.2, 10, 32 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate run as a separate study for laboratory validation.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

13 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.8 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.8 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.26, 1.7 and 2.8 mg/L, however cell debris was observed to be present in the test cultures at 18 and 34 mg/L.

Results with reference substance (positive control):

The positive control (run as separate study), potassium dichromate, gave an ErC50 (0 to 72 hrs) = 1.6 mg/L which is within the normal range for the laboratory.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Observations on Test Item Solubility

At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control, 0.26, 1.7 and 2.8 mg/L test cultures were observed to be green dispersions whilst the 18 and 34 mg/L test cultures were observed to be clear colourless solutions.

Validity criteria fulfilled:

yes

Conclusions:

The test substance algae 72-hour ECr50 value was 13 mg/L and the NOEC was 2.8 mg/L.

Executive summary:

Introduction

The study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test".

Method

Following preliminary range-finding test Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.

Due to the volatile nature of the test item, testing was conducted in completely filled, stoppered test vessels in order to minimise possible losses due to volatilisation. In order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Results

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.26 to 34 mg/L (26% to 55% of the nominal test concentrations). Analysis of the test preparations at 72 hours showed measured test concentrations to range from 0.25 to 36 mg/L (91% to 108% of the 0-Hour measured test concentrations) and as such it was considered appropriate to calculate the results based on the 0-Hour measured test concentrations.

It was considered likely that the failure to obtain near nominal concentrations in the 0-Hour test preparations was due to losses through volatilisation during the time taken to prepare and sample the test solutions. Failure to achieve near nominal concentrations was considered to have had no impact on the outcome of the test given that a concentration dose response was observed allowing the calculation of both EC50 and NOEC values at the concentrations achieved.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the 0-Hour measured test concentrations:

Response Variable	EC50 (mg/L)	95% Confidence Limits (mg/L)	No Observed Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	13	11 - 16	2.8	18
Yield	12	-	2.8	18

Description of key information

Toxicity of the test substance to freshwater algae based on growth rate.

Key value for chemical safety assessment

EC50 for freshwater algae:

13 mg/L

EC10 or NOEC for freshwater algae:

2.8 mg/L