Tributyl borate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

other: experimental study on hydrolysis product Boric acid

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

At the start and end of the growth inhibition test, single 50 ml samples were taken from the control series without algae containing nominal test substance concentrations of 0, 32, 100 and 320 mg/L. The samples were placed in a refrigerator until transfer to the analytical sciences Division for analyses.

One sample was taken from each flask after 0, 26.0, 49.5 and 74.5h and the number of algal cells per ml in the samples was analysed.

Vehicle:

no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Source: ATCC 22662, supplied by the 'American Type Culture Collection', 12301 Parklawn Drive, Rockville, Maryland 20852, USA.
- A preculture of algae in the exponential growth phase was prepared as detailed in OECD Guideline no. 201

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

74.5 h

Hardness:

24.2 mg CaCO3/L

Test temperature:

23 +- 2°C

pH:

at test initiation: 7.5-8.3
at end of test: 8.0-8.3

Nominal and measured concentrations:

Nominal concentrations : 0, 32, 56, 100, 181, 321, 562 mg/L boric acid => 0, 5.6, 9.8, 17.5, 31.7, 56.2, 98.4 mg B/L
The measured concentrations were found to be 95.4 -94.7 % of the nominal concentrations, and they show a linear relationship with the nominal concentrations. The test substance also appeared to be stable during the test, and therefore nominal concentrations were used to report the test results according to the EU C.3 Guideline.
The samples were analysed for B using inductively coupled atomic emission spectrometry (ICP-AES) at a wavelength of 249.704 nm. The limit of detection is 0.03 mg/L, the limit of determination 0.10 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 200 ml conical test flasks covered with silicone sponge caps
- Initial cells density: 10^6 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: OECD guideline medium (1984) with 150 mg/L NaHCO3 instead of 50 mg/L and addition of Fe-citrate


OTHER TEST CONDITIONS
- Light intensity and quality: fluorescent lamps within standard of 60-120 µmol/(s.m²)



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter Multisizer IIe


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0, 0.10, 1.0, 10, 32.8, 102, 328 and 1025 mg/L

Duration:

3 d

Dose descriptor:

EC10

Effect conc.:

24.5 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

biomass

Remarks on result:

other: Effect concentration with regard to the area under the growth curves; analytical confirmation of B concentrations; confidence limits: 17.4 - 31.4 mg/L

Duration:

3 d

Dose descriptor:

EC10

Effect conc.:

35 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

growth rate

Remarks on result:

other: Effect concentration with regard to the growth rate during exponential growth; analytical confirmation of B concentrations

Duration:

3 d

Dose descriptor:

NOEC

Effect conc.:

17.5 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

growth rate

Remarks on result:

other: analytical confirmation of B concentrations

Duration:

3 d

Dose descriptor:

EC50

Effect conc.:

40.2 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

biomass

Remarks on result:

other: Effect concentration with regard to the area under the growth curves; analytical confirmation of B concentrations; confidence limits: 31.5-55.9 mg/L

Duration:

3 d

Dose descriptor:

EC50

Effect conc.:

52.4 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

growth rate

Remarks on result:

other: Effect concentration with regard to the growth rate during exponential growth; analytical confirmation of B concentrations; confidence limits: 45.4 - 59.4 mg/L

Details on results:

NOEC and EC10 values are provided for the endpoints reported, i.e. biomass and growth rate. In general preference is given to the use of EC10 values for PNEC derivation. However, for growth rate no confidence limits could be calculated. therefore the NOEC for growth rate was selected. For algae, the endpoint growth rate is preferred over the endpoint biomass.

Reported statistics and error estimates:

The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A.
For the NOEC calculations were carried out using the DEBtox software package according to the Dynamic Energy Budgets Theory developed by Kooijman and Bedeaux. Model parameters for population growth and their asymptotic standard deviation and correlation coefficients were estimated. The NEC was calculated from the profile ln likelihood function.

The tests fulfilled the validity criteria of sufficient growth (control growth rate was 0.054 h-1) and a minimum increase of test medium pH (highest pH was 8.3)

Validity criteria fulfilled:

yes

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

other: experimental study on hydrolysis product Boric acid

Adequacy of study:

key study

Study period:

13 to 16 July 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

Qualifier:

according to guideline

Guideline:

other: ASTM E1218-97a Standard Guide for Conducting Static 96-h Toxicity Tests with Microalgae

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Analytical measurements were made on samples collected at 0 h and 72 h. Samples at 0 h were taken from parent solutions. Samples at 72 h were taken from combined replicate samples. To remove algae, 72 h samples were centrifuged and supernatant removed for analysis

Vehicle:

no

Details on test solutions:

Test medium was synthetic seawater (Marinemix, Wiegandt GmbH) prepared by addition to autoclaved lab reagent water, with a saltwater algal nutrient medium enrichment. Boron concentrations in this medium (geomean 7.7 mg B/L) exceeded the boron levels typically expected in natural seawater (ca 4.5 mg B/L).

Test organisms (species):

Phaeodactylum tricornutum

Details on test organisms:

Stock obtained from University of Texas at Austin (UTEX). Cultures maintained in 14 h light: 10 hr dark at 20 degrees C. Four days prior to initiation of definitive test, new culture was cloned and incubated under continuous illumination of approximately 8000 lux.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

20.5 to 21.0 degrees C

pH:

7.2 to 8.1

Salinity:

30 +/- 2 ppt

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 6.3, 13, 25, 50 and 100 mg B/L (as added boron)
Geometric Mean Measured Total Concentrations: 7.68 (control), 14.7, 23.3, 35.6, 70.1, and 109 mg B/L
Geometric Mean Calculated Added Concentrations: 0 (control), 7.0, 15.6, 27.9, 62.4, 101.3 mg B/L

Details on test conditions:

The exposure flasks were 250-mL Erlenmeyer flasks with foam stoppers. Prior to test initiation, the flasks were cleaned and autoclaved according to ABC standard operating procedures. The control was replicated six times and each test substance treatment was replicated three times. Each replicate contained 100 mL of the appropriate parent solution. An additional replicate (replicate D) of the 6.3 mg B/L test substance treatment, containing 100 mL of the appropriate parent solution, was also prepared and used to evaluate the potential for incorporation of the test substance into the algal biomass. At test initiation, all replicates of the controls and each A, B, and C replicate of each test substance treatment were inoculated with 1.0 mL of an algal concentrate containing approximately 1.0 x 10^6 cells/mL, resulting in a final density of approximately 1.0 x 10^4 cells/mL for each flask. The replicates were inoculated with algae within 30 minutes after test solution preparation. At 24, 48, and 72 hours (±1 hour), cell density was measured by direct microscopic counting with a hemacytometer. Replicate D of the 6.3 mg B/L test substance treatment was not inoculated with algae.
During the three-day exposure period, the flasks were randomly positioned daily using a computer-generated random number table and incubated at 20 ± 2°C in a temperature controlled environmental chamber under continuous cool-white fluorescent lighting. A continuous recording of environmental chamber temperature was made from one uninoculated blank flask using an electronic datalogger with thermistor probe. Light intensity was measured daily with a LI-COR Model LI-189 light meter equipped with a LI-COR photometric sensor and ranged from 8,014 to 8,254 lux. The flasks were swirled on an orbital shaker table at 100 rpm throughout the test. Temperature and pH were measured in all parent solutions prior to distribution of the solutions to the test flasks. At 72 hours, temperature and pH were measured in replicate A of the controls and each test substance treatment. All temperature and pH measurements of the test solutions were performed with a WTW Model pH 330i meter.

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

50.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

growth rate

Remarks on result:

other: added boron (46.0 to 55.4)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

66 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

growth rate

Remarks on result:

other: added boron (63.5 to 67.7)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

27.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

growth rate

Remarks on result:

other: added boron

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

41.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

biomass

Remarks on result:

other: added boron (40.9 to 42.8)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

54 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

biomass

Remarks on result:

other: added boron (52.9 to 55.2)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

27.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

biomass

Remarks on result:

other: added boron

Duration:

62.4 h

Dose descriptor:

LOEC

Effect conc.:

70.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

other: growth rate and biomass

Remarks on result:

other: added boron

Details on results:

Control algal growth met acceptable limits. Cell numbers were greater than 16 times the initial density. Coefficient of variation among control replicates did not exceed 7% and pH did not increase more than 1 unit. All water quality parameters remained within acceptable limits.

Reported statistics and error estimates:

Statistical analysis based on one-way ANOVA followed by Dunnett's test. ECx estimates obtained from probit method and trimmed Spearman-Karber method if poor fit in the probit approach.

Algal Endpoint values

Group	Nominal Concentration (mg B/L)	Geomean measured (mg B/L)	Cell Density 72-h (cells/mL x 10e4)	Mean Growth Rate (cells/mL-h)	Yield (cells/mL x 10e4)
C	0	7.7	42	0.052	40.8
T1	6.3	14.7	45	0.053	43.7
T2	13	23.3	43	0.052	41.7
T3	25	35.6	41	0.052	39.7
T4	50	70.1	9.9*	0.032*	8.9*
T5	100	109.0	1.1*	0.001*	0.1*
* Significant difference from control (p<0.05)

Validity criteria fulfilled:

yes

Conclusions:

Growth inhibition of the marine diatom Phaeodactylum tricornutum exposed to boric acid was measured. Cell densities were measured daily and used to determine growth rate and biomass yield. Based on total boron measured values, NOECs were 35.6 mg B/L and lower, EC10-growth was 58.4 mg B/L, EC50-growth was 73.3 mg B/L, EC10-yield was 49.5 mg B/L, and EC50-yield was 61.7 mg B/L. Based on added boron measured values, NOECs were 27.9 mg B/L and lower, EC10-growth was 50.7 mg B/L, EC50-growth was 65.6 mg B/L, EC10-yield was 41.8 mg B/L, and EC50-yield was 54.0 mg B/L. The test met acceptability criteria for the method, based upon ISO 10253:2006.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

225 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 204 - 246 mg/L

Key result

Duration:

96 h

Dose descriptor:

other: NOAEC

Effect conc.:

129 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Reported statistics and error estimates:

Cell densities, mean cell densities and percent inhibition values were calculated using "The SAS System for Windows", Release 6.12, while statistical analyses were conducted using "TOXSTAT Release 3.5". Percent growth inhibition was calculated for each treatment group as the percent reduction in mean cell density relative to the mean cell density in the control replicates. The following formula was used:
Percent Inhibition = [(Mean Cell Density control - Mean Cell Densitv treatment) / Mean Cell Density control] x 100.
Cell densities were analyzed statistically to estimate the EC 10, EC50 and EC90 values (i.e., the theoretical test concentrations that would produce a 10, 50 or 90% reduction in cell density, respectively) and 95% confidence limits for each test period. The EC values and 95% confidence limits were calculated by linear interpolation. Cell densities were evaluated for normality and homogeneity of variances using the Shapiro-Wilk's test and Bartlett-'s test, respectively. The data failed the assumption of homogeneity and was transformed by log base 10 to correct the condition. The treatment groups were then compared to the control using Dunnett's test. Results of the statistical analyses were used to determine the no-observed-adverse-effect-concentration (NOAEC).

Measurement of Test Concentrations

Nominal concentrations selected for use in this study were 125, 250, 500, 1000 and 2000 mg n-Butyl Alcohol/L. Samples collected at the beginning of the test had measured concentrations that ranged from 97 to 103% of nominal. Samples collected on Day 4 had recoveries that ranged from less than the limit of quantitation (< LOQ) to 73% of nominal. Four of the five test concentrations had dropped below 70% of nominal at exposure termination. Consequently, the test results were calculated based on the Day 0 measured concentrations.

Environmental Measurements

The temperatures ranged from 23.2 to 25.3°C and were within the range established for the test (24±2°C). The average light intensity ranged from 4240 to 4568 lux and was within the desired range for the test (3870 to 4730 lux). Measurements of pH were 7.4 on Day 0 and ranged from 6.8 to 7.7 at 96 hours.

Cell Density Analyses

The effect of n-Butyl Alcohol upon Selenastrum capricornutum was determined by evaluating differences in cell densities. Mean cell densities were used to calculate growth inhibition values for each 24-hour period. Mean cell densities (incl. the Percent inhibition values) and the EC10, EC50 and EC90 values and 95% confidence limits for each 24-hour interval are attached (see 'Attached background material', Tables).

Changes in cell density indicated that exponential growth occurred in the negative control replicates. The coefficient of variation for the control replicates was 8.5%. There were no statistically significant (p>0.05) reductions in cell density at 129 mg n-Butyl Alcohol/L when compared to the negative control group. The 241, 491, 1010 and 1980 mg n-Butyl Alcohol/L treatments exhibited statisticall significant (p<=0.05) reductions in cell density upon comparison to the negative control group. Growth inhibition values for those treatments at 96 hours were 57, 83, 100 and 100%, respectively.

Visual and Microscopic Observations

After 96 hours of exposure, there were no signs of clumping, flocculation or adherence of the algae to the test flasks in the negative control or any n-Butyl Alcohol treatment group. In addition, there were no noticeable changes in cell size, color or morphology when compared to the negative control.

Reversibility of Growth Inhibition

Aliquots of the 1980 mg n-Butyl Alcohol/L treatment group were diluted with algal medium to a concentration of the test substance expected to have no effect on growth (30 mg n-Butyl Alcohol/L). The recovery solution was monitored to determine if the observed effects of the test substance upon algal growth were algistatic or algicidal. Algal cells in the 1980 mg n-Butyl Alcohol/L treatment group resumed normal growth after nine days during the recovery period.

Validity criteria fulfilled:

yes

Conclusions:

- 96-hour EC50 = 225 mg/L (95% CL: 204 - 246 mg/L)
- 96-hour NOAEC = 129 mg/L

Executive summary:

The objective of the study was to evaluate the acute toxicity of n-Butyl Alcohol to the growth of the freshwater alga, Selenastrum capricornutum, during a 96-hour exposure period. The study was conducted according to OECD Guidelines for Testing of Chemicals, 201 and Title 40 of the Code of Federal Regulations, Part 797, Section 1050.

The freshwater alga, Selenastrum capricornutum, was exposed to a geometric series of five test concentrations and a negative (culture medium) control under static conditions for 96 hours. Three replicate test chambers were maintained for each treatment and control group. Nominal test concentrations were selected in consultation with the Sponsor and were based upon the results of a range finding test. The nominal test concentrations selected were 125, 250, 500,1000 and 2000 mg/L.

At test initiation, an inoculum of the algal cells was prepared at a concentration of approximately 1.0 x 10⁶ cells/mL. The concentration of algal cells in the inoculum was verified and 1.0 mL was added to each test chamber to achieve a nominal concentration of approximately 1.0 x 10⁴ cells/mL. Samples were collected from each replicate test chamber at approximately 24-hour intervals during the test to determine cell densities. Cell densities were measured for each replicate and were used to calculate percent inhibition values relative to the control over the 96-hour exposure period. EC10, EC50 and EC90 values were calculated, if possible, based upon cell densities for each 24-hour interval. The no-observed-adverse-effect-concentration (NOAEC) was determined based upon statistical evaluation of the cell densities. At the end of the 96-hour exposure, algistatic effects were differentiated from algicidal effects in the treatment which was maximally inhibited.

The 96-hour EC10 value for Selenastrum capricornutum exposed to n-Butyl Alcohol was 134 mg/L. The 95% confidence limits were 124 and 167 mg/L. The 96-hour EC50 value was 225 mg/L with 95% confidence limits of 204 and 246 mg/L. The 96-hour EC90 value was 717 mg/L with 95% confidence limits of 586 to 809 mg/L. The statistically defined NOAEC was 129 mg/L. Algal cells in the 1980 mg/L treatment resumed normal growth after nine days during the recovery period. Consequently, the effects upon algal growth were considered to be algistatic, rather than algicidal, at the concentrations tested.

Description of key information

Tributyl borate is rapidly hydrolyzed to Boric acid and n-Butanol in the presence of water (18.3 sec at 21°C).

Reliable data of the hydrolysis products n-Butanol and Boric acid are used to address the endpoint, which is entirely appropriate to draw conclusions on the toxicity of Tributyl borate to aquatic algae:

1) n-Butanol - Toxicity to freshwater algae:

- (96h) EC50 for algae (Pseudokirchneriella subcapitata): 225 mg/L (95% CL: 204 - 246 mg/L) [based on: growth rate; OECD 201; GLP];

- (96h) NOAEC for algae (Pseudokirchneriella subcapitata): 129 mg/L [OECD 201; GLP].

2) Boric acid - Toxicity to algae (based on total boron measured values):

a) Freshwater algae

- (72h) EC50 for algae (Pseudokirchneriella subcapitata): 52.4 mg B/L [based on: growth rate; OECD 201; GLP]; EC50 considering the boron content (4.6968%) of Tributyl borate: 1116 mg/L;

- (72h) NOEC for algae (Pseudokirchneriella subcapitata): 17.5 mg B/L [OECD 201; GLP]; NOEC considering the boron content (4.6968%) of Tributyl borate: 373 mg/L.

b) Marine water algae

- (72h) EC50 for algae (Phaeodactylum tricornutum): 66 mg B/L (95% CL: 63.5 - 67.7 mg B/L) [based on: growth rate; ISO 10253 and ASTM E1218-97a; GLP]; EC50 considering the boron content (4.6968%) of Tributyl borate: 1405 mg/L;

- (72h) NOEC for algae (Phaeodactylum tricornutum): 27.9 mg B/L [ISO 10253 and ASTM E1218-97a; GLP]; NOEC considering the boron content (4.6968%) of Tributyl borate: 594 mg/L.