Bicyclo[2.2.1]heptanebis(methylamine)

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:

31 March 2016 to 12 October 2016

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)

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 quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Nominal amounts of test material (200 and 64 mg) were each separately dissolved in culture medium and the volume adjusted to 2 liters to give 100 and 32 mg/L stock solutions respectively. The pH of these stock solutions was measured and adjusted to pH 7.5 prior to performing serial dilutions to give further stock solutions of 10, 3.2 and 1.0 mg/L. An aliquot (1800 mL) of each of the stock solutions was separately inoculated with algal suspension (21.0 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4.
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
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 illumination at 21 ± 2 °C.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C

pH:

7.5

Nominal and measured concentrations:

Range-Finding Test
0.1, 1.0, 10 and 100 mg/L (nominal).

Initial Experiment
6.25, 12.5, 25, 50 and 100 mg/L.

Definitive Test
1.0, 3.2, 10, 32, 100 mg/L (nominal).

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flasks
- Type: Closed; the flasks were sealed with ground glass stoppers
- Material, size, headspace, fill volume: Glass conical flasks.
- Aeration: The flasks were kept under constant agitation by orbital shaker (100 – 150 rpm).
- No. of vessels per concentration: 3
- No. of vessels per control: 6
- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 4.29 x 10^5 cells per mL. Inoculation of 1800 mL of test medium with 21.0 mL of this algal suspension gave an initial nominal cell density of 5 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: The culture medium used for the range-finding test, initial experiment and definitive tests was the same as that used to maintain the stock culture. The media used in the definitive test was prepared with the addition of 250 mg/L of sodium bicarbonate to counteract the increase in pH due to algal growth in an enclosed system.

- Intervals of water quality measurement: The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily.

OTHER TEST CONDITIONS
- Adjustment of pH: Yes. It was noted that the pH of the 100 mg/L test concentration prepared in the range-finding test was relatively high (pH 9.8). As such it was considered appropriate to adjust the pH of the definitive test preparations to 7.5 prior to performing serial dilutions and inoculating with algal cells in order to determine whether it was the alkaline nature of the test item which caused the inhibitory effects observed.
- Photoperiod: Continuous illumination.
- Light intensity and quality: Intensity approximately 7000 lux provided by warm white lighting (380 – 730 nm).

EFFECT PARAMETERS MEASURED
Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

TEST CONCENTRATIONS
- Range finding study
The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of 72 hours.
The test was conducted in 250 mL glass conical flasks each completely filled with test preparation and sealed with ground glass stoppers to reduce evaporation. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture medium.
A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 litre to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (900 mL) of each of the stock solutions was separately inoculated with algal suspension (9.0 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then sealed with ground glass stoppers and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis.
- 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):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

35 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence limits were 33 - 36 mg/L.

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

28 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

RANGE-FINDING TEST
The results showed no effect on growth at the test concentrations of 0.10, 1.0 and 10 mg/L. However, growth was observed to be reduced at 100 mg/L.
Based on this information test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L were selected for the initial experiment.
Chemical analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 94 % to 103 % of nominal indicating that the test item was stable under test conditions.

INITIAL EXPERIMENT
An initial experiment conducted at test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L showed significant inhibition of growth occurred at 6.25 mg/L. It was therefore considered appropriate to conduct the definitive test using a test concentration range of 1.0, 3.2, 10, 32 and 100 mg/L.

DEFINITIVE TEST
- Verification of Test Concentrations
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 86 % to 96 % of nominal and so the results are based on nominal test concentrations only.

- Growth Data
The growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test material over the 72-hour exposure period. As significant effects on growth rate were observed when the pH of the test preparations had been adjusted, it was considered that some other intrinsic property of the test material was causing the inhibitiory effects seen.

- Inhibition of growth rate
The ErC10 (0 - 72 h) was 28 mg/L.
The EcC20 (0 - 72 h) was 31 mg/L.
The ErC50 (0 - 72 h) was 35 mg/L; 95 % confidence limits of 33 - 36 mg/L.
Where ErCx is the test concentration that reduced growth rate by x %.

There were no statistically significant differences (P≥0.05), between the control, 1.0, 3.2 and 10 mg/L test concentrations however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 10 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 32 mg/L.

- Inhibition of Yield
The EyC10 (0 - 72 h) was 6.7 mg/L.
The EyC20 (0 - 72 h) was 9.9 mg/L.
The EyC50 was 20 mg/L; 95 % confidence limits of 16 - 25 mg/L.
Where:
EyCx is the test concentration that reduced yield by x %.

There were no statistically significant differences (P≥0.05), between the control, 1.0 and 3.2 mg/L test concentrations however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 3.2 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 10 mg/L.

- Observations on Cultures
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 1.0, 3.2, 10 and 32 mg/L, however no intact cells were observed to be present in the test cultures at 100 mg/L.

- Observations on Test Material Solubility
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-hour test period all control, 1.0 and 3.2 mg/L test cultures were observed to be green dispersions. The 10 mg/L test cultures were pale green dispersions; the 32 mg/L test cultures were extremely pale green dispersions whilst the 100 mg/L test cultures were clear colorless solutions.

Results with reference substance (positive control):

A positive control (Envigo Study Number MG09PL) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 – 72 h): 1.5 mg/L; 95 % confidence limits 1.3 – 1.7 mg/L
EyC50 (0 – 72 h): 0.79 mg/L; 95 % confidence limits 0.70 – 0.89 mg/L
No Observed Effect Concentration (NOEC) based on growth rate: 0.50 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 1.0 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 1.0 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

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).

Water Quality Criteria

Temperature was maintained at 24 ± 1 ºC throughout the test.

The pH value of the control cultures was observed to increase from pH 7.9 at 0 hours to pH 9.4 at 72 hours. The pH deviation in the control cultures was equal to 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 62 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours: 5.00 x 10^3 cells per mL

Mean cell density of control at 72 hours: 3.11 x 10^5 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 30 % and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35 %.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 2 % and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7 %.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 72-hour ErC50 was determined to be 35 mg/L with 95 % confidence limits of 33 - 36 mg/L, while the 72-hour ErC10 was determined to be 28 mg/L. The No Observed Effect Concentration was 10 mg/L.

Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 201 and EU Method C.3.

Following a preliminary range-finding test and initial experiment, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test material at 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 alkaline nature of the test material, the pH of the 100 and 32 mg/L test preparations was measured and adjusted to pH 7.5 prior to performing serial dilutions and inoculating with algal cells at the start of the test.

Due to the potentially volatile nature of the test material, testing was conducted in completely filled, stoppered test vessels in order to minimise possible losses due to volatilization. Following the recommendations of published data (Herman et al 1990) 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.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 86 % to 96 % of nominal and so the results are based on nominal test concentrations only.

The 72 hour EC50 for growth rate was 35 mg/L with 95 % confidence limits of 33 - 36 mg/L. The NOEC was 10 mg/L and the LOEC was 32 mg/L.

Under the conditions of the study, the 72-hour ErC50 was determined to be 35 mg/L while the 72-hour ErC10 was determined to be 28 mg/L. The No Observed Effect Concentration in terms of growth rate was 10 mg/L.

Description of key information

72 -hour ErC50 35.0 mg/L; 72 -hour ErC10 28.0 mg/L; 72 -hour NOECr 10.0 mg/L (Pseudokirchneriella subcapitata), OECD 201, C.3, H Vryenhoef (2016).

Key value for chemical safety assessment

EC50 for freshwater algae:

35 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L

Additional information

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 201 and EU Method C.3.

Following a preliminary range-finding test and initial experiment, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test material at 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 alkaline nature of the test material, the pH of the 100 and 32 mg/L test preparations was measured and adjusted to pH 7.5 prior to performing serial dilutions and inoculating with algal cells at the start of the test.

Due to the potentially volatile nature of the test material, testing was conducted in completely filled, stoppered test vessels in order to minimise possible losses due to volatilization. Following the recommendations of published data (Herman et al 1990) 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.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 86 % to 96 % of nominal and so the results are based on nominal test concentrations only.

The 72 hour EC50 for growth rate was 35 mg/L with 95 % confidence limits of 33 - 36 mg/L. The NOEC was 10 mg/L and the LOEC was 32 mg/L.

Under the conditions of the study, the 72-hour ErC50 was determined to be 35 mg/L while the 72-hour ErC10 was determined to be 28 mg/L. The No Observed Effect Concentration in terms of growth rate was 10 mg/L.