2,2'-butyliminodiethanol

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:

2015-06-02 - 2015-06-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline Study in compliance with GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material:
not applicable

Analytical monitoring:

yes

Details on sampling:

- Sampling: The test samples were thawed with the aid of sonication. The test samples (72-Hour) were centrifuged at 4500 rpm for 10 minutes in glass tubes, prior to dilution, to remove algal cells. The samples were stored frozen prior to analysis.

Vehicle:

no

Details on test solutions:

Preparation of Calibration Standards
The test item (nominal 100 mg) was dissolved in water (100 mL) to prepare a stock solution with a concentration of 1000 mg/L. This stock solution was further diluted with test medium to produce a solution of 10 mg/L. Defined volumes of this solution were then diluted with test medium to obtain calibration standards in the range of 0.5 to 1.0 mg/L. A second series of calibration standards was similarly prepared in the range of 0.01 to 0.75 mg/L. These solutions were used to determine the recovery and test sample concentrations.

Preparation of Linearity Standards
The test item (nominal 100 mg) was dissolved in water (100 mL) to prepare a stock solution with a concentration of 1000 mg/L. This stock solution was further diluted with test medium to produce a solution of 10 mg/L. Defined volumes of this solution were then diluted with test medium to obtain calibration standards in the range of 0.5 to 1.0 mg/L. A second series of calibration standards was similarly prepared in the range of 0.01 to 0.75 mg/L. These standards were used to evaluate the linearity of the analytical system.

Preparation of Spiked Recovery Samples
To demonstrate the validity of the analytical procedure, volumes of test medium were spiked with the test item and the recovery was assessed. The test item (nominal 100 mg) was initially dissolved in test medium to prepare a stock solution with a concentration of 1000 mg/L. A defined volume of this stock solution was diluted with test medium to obtain spiked recovery samples at a concentration of 100 mg/L. Five replicates at this concentration level were prepared and subjected to the same treatment as the test samples. In addition, test medium without the addition of the test item (synthetic control) was also analyzed.

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 (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland

ACCLIMATION
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100- 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 - 10^5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

not reported

Hardness:

not reported

Test temperature:

24 ± 1 °C

pH:

7.0 - 9.5

Dissolved oxygen:

not reported

Salinity:

not applicable

Nominal and measured concentrations:

Range-finding study:
Nominal (0.1, 1.0, 10 and 100 mg/L) and Measured (0.994, 10.2 and 104 mg/L at 0h; 1.05, 9.63 and 103 mg/L at 72h)

Definitive study:
Nominal (6.25, 12.5, 25, 50 and 100 mg/L) and Measured (6.45, 13.1, 25.9, 51.2 and 105/100 mg/L at 0h; 5.77, 11.6, 23.3, 46.4 and 98.0/95.0 mg/L at 72h)

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flasks
- Type (delete if not applicable): closed (The flasks were plugged with polyurethane foam stoppers.)
- Material, size, headspace, fill volume: glass, 250 mL volume
- Aeration: constantly shaken at approximately 150 rpm for 72 hours
- Initial cells density: initial nominal cell density of 5 x 10E3 cells per mL
- Control end cells density: Mean cell density of control at 72 hours : 4.76 x 10E5 cells per mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes (AAP-medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture medium was prepared using reverse osmosis purified deionized water (Elga Optima 15+ or Elga Purelab Option R-15 BP). Stock solutions were diluted with test medium.
- Culture medium different from test medium: no
- Intervals of water quality measurement: initiation of the test and after 72 hours exposure.

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH: yes (for 100 mg/L)
- Photoperiod: continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 - 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10 for range finding study, 2 for definitive test

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: 0 (control), 6.25, 12.5, 25, 50 and 100 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 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.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

57

95% CI:

>= 51 - <= 56

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL: 51 - 56 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

other: ErC10

Effect conc.:

48 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes

Results with reference substance (positive control):

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) : 1.2 mg/L; 95 % confidence limits 1.1 - 1.4 mg/L
EyC50 (0 - 72 h) : 0.63 mg/L; 95 % confidence limits 0.57 - 0.70 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.50 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 1.0 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 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:

Inhibition of growth rate
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using 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). There were no statistically significant differences (P > 0.05), between the control, 6.25, 12.5 and 25 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 25 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 50 mg/L.

Inhibition of yield
Statistical analysis of the yield data was carried out as in Section 4.2.2.1. There were no statistically significant differences (P>0.05), between the control, 6.25, 12.5 and 25 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 25 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 50 mg/L.

Range-finding Test

The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1. The results showed no effect on growth rate 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 definitive test. A concentration dependent increase in pH was observed in the range-finding test samples at 0 hours in the range of pH 7.0 at 0.10 mg/L through to pH 9.4 at 100 mg/L. At the request of the Sponsor, in order to determine whether it was the alkaline nature of the test item which was causing the inhibition of growth observed, an additional 100 mg/L test preparation was employed in the definitive test which had been pH adjusted to 7.5 prior to inoculation with algal cells. Chemical analysis of the test preparations at 0 and 72 hours showed near nominal test concentrations were obtained indicating that the test item was stable under test conditions.

 

Definitive Test

Verification of Test Concentrations

Analysis of the test preparations at 0 and 72 hours (see Appendix 4) showed measured test concentrations to range from 92 % to 105 % of nominal and hence the results are based on nominal test concentrations only.

 

Growth Data

Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4. The mean cell densities versus time for the definitive test are presented in Figure 1. Percentage inhibition values are plotted against test concentration in Figure 2 and Figure 3. From the data given in Table 2 and Table 4, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period. The data confirmed that whilst the adjustment of the pH of the 100 mg/L test preparation did result in a decrease in the percentage inhibition observed, this decrease was not significant enough to attribute the effects seen in the non-pH adjusted preparations to the alkaline nature of the test item alone. Given that the alkalinity of the test preparations was considered to be due to an intrinsic property of the test item, and that inhibition of growth was also observed in a test preparation where the pH had been adjusted, it was considered appropriate to calculate the results based on the non-pH adjusted test preparations only. Accordingly the following results were determined from the data:

 

Inhibition of Growth Rate

ErC10 (0-72h): 48 mg/L

ErC20 (0 - 72h) : 69 mg/L

ErC50 (0-72h) : >100 mg/L*

 

where ErCx is the test concentration that reduced growth rate by x %.

It was not possible to calculate an ErC50 value as no concentration tested resulted in greater than 50 % inhibition of growth rate.

 

* It was not possible to calculate 95 % confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.

 

Inhibition of Yield

EyC10 (0 -72h): 20 mg/L

EyC20 (0 -72h): 30 mg/L

EyC50 (0 - 72 h): 57 mg/L; 95 % confidence limits 51-65 mg/L

 

Where: EyCx is the test concentration that reduced yield by x %.

 

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 110 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 : 4.30 x E3 cells per mL

Mean cell density of control at 72 hours : 4.76 x E5 cells per mL

 

The mean coefficient of variation for section by section specific growth rate for the control cultures was 25% 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 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7 %.

 

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 6.25, 12.5, 25 and 50 mg/L. Whilst no intact cells were observed to be present in the 100 mg/L test cultures, a little cell debris was observed in the 100 mg/L pH-adjusted test cultures.

 

Water Quality Criteria

The pH values of the control and each test preparation are given in Table 2. Temperature was maintained at 24 ± 1 °C throughout the test. The pH value of the control cultures (see Table 2) was observed to increase from pH 7.0 at 0 hours to pH 7.4 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

 

Observations on Test Item 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, 6.25, 12.5 and 25 mg/L test cultures were observed to be green dispersions whilst the 50 mg/L test cultures were observed to be pale green dispersions. The 100 mg/L test cultures were observed to be extremely pale green dispersions whilst the 100 mg/L pH adjusted test cultures were observed to be pale green dispersions.

Validity criteria fulfilled:

yes

Conclusions:

The ErC50 (72 h) value amounts to > 100 mg/L while the NOErC (72 h) is 25 mg/L.

Executive summary:

The toxicity of the substance BDEA (CAS 102-79-4) towards green algae (Pseudokirchneriella subcapitata) was determined according to OECD Guideline 201 in compliance with GLP.

Following a preliminary range-finding test, the test organisms were exposed to an aqueous solution of the test item at concentrations of 6.25, 12.5, 25, 50 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item formed an alkaline solution when dissolved in culture medium, particularly at the maximum concentration employed of 100 mg/L (pH 9.5). At the request of the Sponsor, in order to determine whether it was the alkaline nature of the test item which gave rise to the observed inhibition, an additional 100 mg/L test concentration was prepared whereby the pH was adjusted to 7.5 prior to inoculation with algae (three replicates). 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 92 % to 105 % of nominal and so the results are based on nominal test concentrations only. The biological data obtained confirmed that whilst the adjustment of the pH of the 100 mg/L test preparation did result in a decrease in the percentage inhibition observed, this decrease was not significant enough to attribute the effects seen in the non-pH adjusted preparations to the alkaline nature of the test item alone. Given that the alkalinity of the test preparations was considered to be due to an intrinsic property of the test item, and that inhibition of growth was also observed in a test preparation where the pH had been adjusted, it was considered appropriate to calculate the results based on the non-pH adjusted test preparations only. Exposure of Pseudokirchneriella subcapitata to the test item gave an ErC50 (72 h) value of > 100 mg/L, ErC10 (72 h) of 48 mg/L, a NOErC (72 h) of 25 mg/L, an EyC50 (72 h) of 57 and a NOEyC of 25 mg/L.

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

Description of key information

Butyldiethanolamine_OECD 202: ErC50 (72 h) > 100 mg/L; NOErC (72 h) = 25 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

25 mg/L

Additional information

The toxicity of the substance BDEA (CAS 102-79-4) towards green algae (Pseudokirchneriella subcapitata) was determined according to OECD Guideline 201 in compliance with GLP.

Following a preliminary range-finding test, the test organisms were exposed to an aqueous solution of the test item at concentrations of 6.25, 12.5, 25, 50 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item formed an alkaline solution when dissolved in culture medium, particularly at the maximum concentration employed of 100 mg/L (pH 9.5). At the request of the Sponsor, in order to determine whether it was the alkaline nature of the test item which gave rise to the observed inhibition, an additional 100 mg/L test concentration was prepared whereby the pH was adjusted to 7.5 prior to inoculation with algae (three replicates). 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 92 % to 105 % of nominal and so the results are based on nominal test concentrations only. The biological data obtained confirmed that whilst the adjustment of the pH of the 100 mg/L test preparation did result in a decrease in the percentage inhibition observed, this decrease was not significant enough to attribute the effects seen in the non-pH adjusted preparations to the alkaline nature of the test item alone. Given that the alkalinity of the test preparations was considered to be due to an intrinsic property of the test item, and that inhibition of growth was also observed in a test preparation where the pH had been adjusted, it was considered appropriate to calculate the results based on the non-pH adjusted test preparations only. Exposure of Pseudokirchneriella subcapitata to the test item gave an ErC50 (72 h) value of > 100 mg/L, ErC10 (72 h) of 48 mg/L, a NOErC (72 h) of 25 mg/L, an EyC50 (72 h) of 57 and a NOEyC of 25 mg/L. The results from the positive control with potassium dichromate were within the normal ranges for this reference item.