Diboron trioxide

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well performed study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Test animals were exposed to 10 - 14 concentrations of boric acid and borax at 2 different water hardness levels. Mortality was recorded and LC1 and LC50 calculated. Dyer (2001), calculated the LC10, LC20, LC50 from the original data

GLP compliance:

not specified

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Carassius auratus

Details on test organisms:

TEST ORGANISM
- Common name: Goldfish
- Source: Gravid fish and amphibians were obtained from State and Federal hatcheries at Frankfort, Kentucky, Erwin, Tennessee, and Senecaville, Ohio, and from selected sites within Kentucky.


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: collected from natural spawn

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Hardness:

Hardness 50 : 54.4 mg/L
Hardness 200 : 207.5 mg/L

Test temperature:

24.8 - 27°C

pH:

Hardness 50 : 7.9
Hardness 200 : 7.6

Dissolved oxygen:

ca. 10 mg/L

Nominal and measured concentrations:

Nominal concentrations : 0.05, 0.10, 0.50, 1.00, 5.00, 7.50, 10.00, 25.00, 50.00, 100.00, 200.00, 300.00 mg B/L
Measured concentrations for test with Hardness 50 : 0.05, 0.1, 0.49, 0.9, 5.2, 7, 9.2, 22.5, 48.7, 108, 188.7, 288 mg B/L
Measured concentrations for test with Hardness 200 : 0.05, 0.12, 0.47, 0.9, 4.5, 6.8, 8.33, 32, 51.3, 96.7, 191, 290 mg B/L

Details on test conditions:

TEST SYSTEM
Fish and amphibian eggs were cultured through 4 days posthatching in Pyrex chambers, through which test water was perfused at prescribed flow rates. The toxicant was administered to a mixing chamber situated ahead of each culture dish, using graduated flow from a syringe pump. Synthetic culture water was delivered to the mixing chamber by regulated flow from a peristaltic pump. Flow rates from both syringe and peristaltic pumps were monitored by liquid flow meters. Flow rate was set at 200 ml /hr for 300 ml test chambers. Syringe and peristaltic pumps were provided with calibrated, continuously variable speed controls. The concentration of toxicant delivered to the mixing chamber was regulated by adjusting the mixing ratio from the pumping units and/or by varying the concentration of toxicant delivered from the syringe pump. Solutions from the two channels were mixed with a Teflon-coated magnetic stirring bar, and delivered to the culture chamber under positive pressure.
The system was operated using Brinkmann (model 131900) and Gilson (model HP8) multichannel peristaltic pumps and Sage syringe pumps (model 355)). The latter was fitted with a modified syringe holder, and operated using 10 ml double-ground, glass syringes. Syringes were selected for equal stroke volume, and peristaltic pump channels were fitted with tubing of matched diameters.
For test concentrations of 10 ppm or more, boron compounds generally were added directly to the culture water in the peristaltic pump reservoir, eliminating the need for the syringe pump channel. In such instances, the toxicants remained stable at selected test concentrations for 24 hours or more in polyethylene containers, and important test parameters of culture water were not altered upon standing.
All aquatic bioassay cultures were maintained in walk-in environmental rooms. Culture water was given continuous aeration in the peristaltic pump reservoirs.



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Culture water was prepared from distilled, double deionized water, having a conductivity of 0.25 umhos or less. Routine monitoring was conducted for background contaminants, including mercury, cadmium, lead and other trace metals. Reagent grade calcium, magnesium, sodium and potassium salts were added. The basic stock was prepared to give a water hardness level of 200 ppm (as CaCO3) and a pH of 7.5 - 8.0. Different levels of hardness were achieved by dilution of the basic culture water.
- Chlorine: 98.2 mg/L
- Alkalinity: 82.0 mg/L
- Conductivity: 300 umhos
- Intervals of water quality measurement: daily intervals


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

Duration:

7 d

Dose descriptor:

LC10

Effect conc.:

20.9 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Remarks:

+ teratogenicity

Remarks on result:

other: Hardness 50; confidence limits: 15.5 - 28.0 mg/L

Duration:

7 d

Dose descriptor:

LC10

Effect conc.:

28.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Remarks:

+ teratogenicity

Remarks on result:

other: Hardness 200; confidence limits: 24.4 - 33.6 mg/L

Reported statistics and error estimates:

For Birge & Black : Log probit analysis was used to statistically determine LC01 and LC50 values. Analysis of variance and the t-test were used to determine statistical significance of differences between boric acid and borax toxicity, water hardness levels and other test variables.
The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A.

Reference 2

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

The actual concentrations of Boric Acid, Manufacturing Grade in the test solutions were determined by chemical analysis. Every week samples of ca. 50 ml were taken from the dilution water control and from the exposure media of 0.56, 5.6 and 18 mg B/L. The samples were taken in polyethylene bottles and stored in the refrigerator until transfer (cooled) to the analytical laboratories in Zeist.

Vehicle:

no

Details on test solutions:

Reconstituted freshwater "DSWL-E" prepared from groundwater and salts. Boron concentration in DSWL-E was 0.56 mg B/L. This value was subtracted from all exposure groups to compare nominal with measured concentrations. Results are thus reported as "added" boron.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Source: Eggs were obtained from the commercial fish hatchery Atlanta in Hellevoetsluis, The Netherlands. The stage of embryonic development at the start of the test was checked under a microscope to be the young blastula stage.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

34 d

Hardness:

212 mg/L

Test temperature:

24.1 - 25.7 °C

pH:

7.2 - 8.0

Dissolved oxygen:

> 7.1 mg/L

Nominal and measured concentrations:

Nominal concentrations: Control (0), 0.18, 0.56, 1.8, 5.6, 18, 56 mg B/L
Measured concentrations: Control (0.53), 0.72, 1.11, 2.4, 6.4, 19, 59 mg B/L
When the concentration in the DWSL-E control media was subtracted from measured concentrations, the measured boron concentrations were between 101% and 104% of the nominal values in the newly prepared test solutions and between 102 and 107% in the spent solutions. This is within +- 20% of the nominal concentrations; according to the guideline it is then allowed to base the results of the test on nominal values. Results are therefore reported as "added" boron.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 1 L glass beakers, each containing 800 ml of test solution or control medium, covered with a watch glass
- Aeration: slightly aerated
- Renewal rate of test solution (frequency/flow rate): Every Monday, Wednesday and Friday
- No. of fertilized eggs/embryos per vessel: 45 reduced to 20 after 24h exposure
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: DSWL-E, prepared from ground water and salts
- Total organic carbon: 1.6 mg/L
- Chlorine: 3.5 mmol/L
- Ca/mg ratio: 2 : 1
- Culture medium different from test medium: No
- Intervals of water quality measurement:pH values, oxygen conc. and temp. were measured in all test vessels at the beginning and at the end of the test, as well as once per week in the freshly prepared and in the spent media.


OTHER TEST CONDITIONS
- Photoperiod: 16h light : 8h dark with a transition period of 30 min



RANGE-FINDING STUDY
- Appropriate concentrations were determined in a semi static preliminary range finding test. Six concentrations of Boric Acid, Manufacturing Grade were tested. DSWL-E was used as a control.

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

6.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

6.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

length

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

6.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

weight

Duration:

34 d

Dose descriptor:

LC10

Effect conc.:

18.3 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Remarks on result:

other: confidence limits: 14.0-24.1 mg/L

Duration:

34 d

Dose descriptor:

EC10

Effect conc.:

18 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

length

Remarks on result:

other: confidence limits: 9.8-33.1 mg/L

Duration:

34 d

Dose descriptor:

EC10

Effect conc.:

6.9 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

weight

Remarks on result:

other: confidence limits: 2.0-24.1 mg/L

Details on results:

The study authors considered the mean dry weight for Dose 1 (0.18 mg B/L nominal concentration) to be an outlier but provided no explanation for why. Data from that group were used in evaluation of all other endpoints. No procedural deviations for the dose 1 replicates were noted that might technically justify why those data were not valid. The observations for dry weight were within the range observed for other replicates. The observation of unexpected data does not provide, by itself, a justification for exclusion of the data. Consequently, all data from dose 1 replicates needs to be included in the analysis of the results.

Reported statistics and error estimates:

The study authors determined statistical significance for mortality with a binomial test at the 95 % significance level, combining the results of the quadruplicates. The study authors reported statistical significance for growth based on the two-tailed Dunnett-test with a 95 % and 99 % significance level. In both cases the observations at each concentration were compared with those of the control. An independent statistician was asked to review the study to determine if the authors used the most appropriate statistics, based on the properties of the data set and whether other approaches might be justified. The OECD Guideline 210 does not specify which statistical techniques may be used to evaluate the data, other than they should be appropriate for use on the data. Dunnett's t-test is suggested, which compares only the control means with the means of the other exposure groups. However, the Guideline recommends that the sponsor consult with a statistician for analysis of the data. More recent OECD statistical guidance (2006) delineates some appropriate procedures for fish early life stage data analysis. Using multiple statistical approaches, the data for egg hatch, mortality and fish length are consistent in showing statistical effects at exposures of 18 mg B/L and above. However, statistical evaluations of fish dry weight were inconstent, as described below, regarding whether the LOEC for dry weight was 18.0 mg B/L, 5.6 mg B/L or 0.18 mg B/L. The exposures ranging from 0.18 to 5.6 mg B/L were not statistically different from each other. The statistical review concluded that the 5.6 mg B/L group should be considered to be the NOEC based on multiple statistical approaches. This is consistent with the conclusions written by thestudy authors. The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A

Mean values for treatment groups are shown below:

Data Values	Control	Dose 1	Dose 2	Dose 3	Dose 4	Dose 5	Dose 6
% eggs hatched after 6 days	100%	100%	100%	100%	100%	100%	82%
% mortality after 34 d	0%	1.1%	0%	2.5%	0%	15%	100%
Length (cm)	1.34	1.33	1.36	1.39	1.34	1.13	-
+/-(std dev)	(0.17)	(0.22)	(0.19)	(0.18)	(0.20)	(0.13)	-
Dry Weight (mg)	3.40	2.77	2.91	3.19	2.62	1.48	-
+/- std dev	(0.11)	(0.18)	(0.41)	(0.34)	(0.22)	(0.44)	-

The statistical conclusions as reported by the study authors are shown below:

34 d	NOEC	5.6 mg/L	nominal	element	mortality
34 d	LOEC	18 mg/L	nominal	element	mortality
34 d	NOEC	5.6 mg/L	nominal	element	other: Condition
34 d	LOEC	18 mg/L	nominal	element	other: Condition
34 d	NOEC	5.6 mg/L	nominal	element	length
34 d	LOEC	18 mg/L	nominal	element	length
34 d	NOEC	5.6 mg/L	nominal	element	dry weight
34 d	LOEC	18 mg/L	nominal	element	dry weight

Reference 3

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well performed study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Test animals were exposed to 10 - 14 concentrations of boric acid at 2 different water hardness levels

GLP compliance:

not specified

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Micropterus salmoides

Details on test organisms:

TEST ORGANISM
- Common name: Largemouth bass
- Source: Frankfort National Fish Hatchery, Frankfort, Kentucky


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: collected from natural spawn

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

11 d

Hardness:

204 (190-225) mg/L

Test temperature:

20.0 (20.0-21.0) °C

pH:

7.5 (7.0-7.7)

Dissolved oxygen:

8.4 (7.8-8.9) mg/L

Nominal and measured concentrations:

Nominal concentrations : 0.05, 0.10, 0.50, 1.00, 5.00, 7.50, 10.00, 25.00, 50.00, 100.00, 200.00, 300.00 mg B/L
Measured concentrations for test with Hardness 200 : 0.009, 0.109, 1.39, 12.17, 25.08, 51.13, 101.4, 193.1, 301.7, 396.5, 491.5 mg B/L

Details on test conditions:

TEST SYSTEM
Fish and amphibian eggs were cultured through 4 days posthatching in Pyrex chambers, through which test water was perfused at prescribed flow rates. The toxicant was administered to a mixing chamber situated ahead of each culture dish, using graduated flow from a syringe pump. Synthetic culture water was delivered to the mixing chamber by regulated flow from a peristaltic pump. Flow rates from both syringe and peristaltic pumps were monitored by liquid flow meters. Flow rate was set at 200 ml /hr for 300 ml test chambers. Syringe and peristaltic pumps were provided with calibrated, continuously variable speed controls. The concentration of toxicant delivered to the mixing chamber was regulated by adjusting the mixing ratio from the pumping units and/or by varying the concentration of toxicant delivered from the syringe pump. Solutions from the two channels were mixed with a Teflon-coated magnetic stirring bar, and delivered to the culture chamber under positive pressure.
The system was operated using Brinkmann (model 131900) and Gilson (model HP8) multichannel peristaltic pumps and Sage syringe pumps (model 355)). The latter was fitted with a modified syringe holder, and operated using 10 ml double-ground, glass syringes. Syringes were selected for equal stroke volume, and peristaltic pump channels were fitted with tubing of matched diameters.
For test concentrations of 10 ppm or more, boron compounds generally were added directly to the culture water in the peristaltic pump reservoir, eliminating the need for the syringe pump channel. In such instances, the toxicants remained stable at selected test concentrations for 24 hours or more in polyethylene containers, and important test parameters of culture water were not altered upon standing.
All aquatic bioassay cultures were maintained in walk-in environmental rooms. Culture water was given continuous aeration in the peristaltic pump reservoirs.



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Culture water was prepared from distilled, double deionized water, having a conductivity of 0.25 umhos or less. Routine monitoring was conducted for background contaminants, including mercury, cadmium, lead and other trace metals. Reagent grade calcium, magnesium, sodium and potassium salts were added. The basic stock was prepared to give a water hardness level of 200 ppm (as CaCO3) and a pH of 7.5 - 8.0. Different levels of hardness were achieved by dilution of the basic culture water.
- Chlorine: 98.2 mg/L
- Alkalinity: 65.3 mg/L
- Conductivity: 282 umhos
- Intervals of water quality measurement: daily intervals

Duration:

11 d

Dose descriptor:

LC10

Effect conc.:

36.8 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Remarks on result:

other: confidence limits: 22.6 - 59.4 mg/L

Reported statistics and error estimates:

For Birge & Black : Log probit analysis was used to statistically determine LC01 and LC50 values. Analysis of variance and the t-test were used to determine statistical significance of differences between boric acid and borax toxicity, water hardness levels and other test variables.
The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A.

Reference 4

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well performed study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Test animals were exposed to 10 - 14 concentrations of boric acid and borax at 2 different water hardness levels.

GLP compliance:

no

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Ictalurus punctatus

Details on test organisms:

TEST ORGANISM
- Common name: Channel catfish
- Source: Gravid fish and amphibians were obtained from State and Federal hatcheries at Frankfort, Kentucky, Erwin, Tennessee, and Senecaville, Ohio, and from selected sites within Kentucky.


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: collected from natural spawn

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

9 d

Hardness:

Hardness 50 : 51.8 mg/L
Hardness 200 : 212 mg/L

Test temperature:

24.7 - 29.4°C

pH:

Hardness 50 : 7.5
Hardness 200 : 7.6

Dissolved oxygen:

ca. 10 mg/L

Nominal and measured concentrations:

Nominal concentrations : 0.05, 0.10, 0.50, 1.00, 5.00, 7.50, 10.00, 25.00, 50.00, 100.00, 200.00, 300.00 mg B/L
Measured concentrations for test with Hardness 50 : 0.01, 0.05, 0.11, 0.49, 1.01, 5.42, 7.43, 10, 24.9, 51.4, 98.3, 151, 177, 306.4 mg B/L
Measured concentrations for test with Hardness 200 : 0.01, 0.05, 0.53, 0.77, 0.96, 2.33, 4.9, 7.4, 9.43, 25.1, 48.3, 77.7, 140, 302 mg B/L

Details on test conditions:

TEST SYSTEM
Fish and amphibian eggs were cultured through 4 days posthatching in Pyrex chambers, through which test water was perfused at prescribed flow rates. The toxicant was administered to a mixing chamber situated ahead of each culture dish, using graduated flow from a syringe pump. Synthetic culture water was delivered to the mixing chamber by regulated flow from a peristaltic pump. Flow rates from both syringe and peristaltic pumps were monitored by liquid flow meters. Flow rate was set at 200 ml /hr for 300 ml test chambers. Syringe and peristaltic pumps were provided with calibrated, continuously variable speed controls. The concentration of toxicant delivered to the mixing chamber was regulated by adjusting the mixing ratio from the pumping units and/or by varying the concentration of toxicant delivered from the syringe pump. Solutions from the two channels were mixed with a Teflon-coated magnetic stirring bar, and delivered to the culture chamber under positive pressure.
The system was operated using Brinkmann (model 131900) and Gilson (model HP8) multichannel peristaltic pumps and Sage syringe pumps (model 355)). The latter was fitted with a modified syringe holder, and operated using 10 ml double-ground, glass syringes. Syringes were selected for equal stroke volume, and peristaltic pump channels were fitted with tubing of matched diameters.
For test concentrations of 10 ppm or more, boron compounds generally were added directly to the culture water in the peristaltic pump reservoir, eliminating the need for the syringe pump channel. In such instances, the toxicants remained stable at selected test concentrations for 24 hours or more in polyethylene containers, and important test parameters of culture water were not altered upon standing.
All aquatic bioassay cultures were maintained in walk-in environmental rooms. Culture water was given continuous aeration in the peristaltic pump reservoirs.



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Culture water was prepared from distilled, double deionized water, having a conductivity of 0.25 umhos or less. Routine monitoring was conducted for background contaminants, including mercury, cadmium, lead and other trace metals. Reagent grade calcium, magnesium, sodium and potassium salts were added. The basic stock was prepared to give a water hardness level of 200 ppm (as CaCO3) and a pH of 7.5 - 8.0. Different levels of hardness were achieved by dilution of the basic culture water.
- Chlorine: 98.2 mg/L
- Alkalinity: 82.0 mg/L
- Conductivity: 300 umhos
- Intervals of water quality measurement: daily intervals


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

Duration:

9 d

Dose descriptor:

LC10

Effect conc.:

11.9 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Remarks:

+ teratogenicity

Remarks on result:

other: Hardness 50; confidence lilmits: 0.2 - 859 mg/L

Duration:

9 d

Dose descriptor:

LC10

Effect conc.:

3.5 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Remarks:

+ teratogenicity

Remarks on result:

other: Hardness 200; confidence limits: 2.4 - 5.1 mg/L

Reported statistics and error estimates:

For Birge & Black : Log probit analysis was used to statistically determine LC01 and LC50 values. Analysis of variance and the t-test were used to determine statistical significance of differences between boric acid and borax toxicity, water hardness levels and other test variables.
The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A.

Reference 5

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well perfofrmed study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Test animals were exposed to 10 - 14 concentrations of boric acid and borax at 2 different water hardness levels.

GLP compliance:

not specified

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Ictalurus punctatus

Details on test organisms:

TEST ORGANISM
- Common name: Channel catfish
- Source: Gravid fish and amphibians were obtained from State and Federal hatcheries at Frankfort, Kentucky, Erwin, Tennessee, and Senecaville, Ohio, and from selected sites within Kentucky.


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: collected from natural spawn

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

9 d

Hardness:

Hardness 50 : 46.7 mg/L
Hardness 200 : 194.7 mg/L

Test temperature:

24.7 - 29.4°C

pH:

Hardness 50 : 8.5
Hardness 200 : 8.2

Dissolved oxygen:

ca. 10 mg/L

Nominal and measured concentrations:

Nominal concentrations : 0.05, 0.10, 0.50, 1.00, 5.00, 7.50, 10.00, 25.00, 50.00, 100.00, 200.00, 300.00 mg B/L
Measured concentrations for test with Hardness 50 : 0.05, 0.45, 0.92, 4.56, 7.21, 9, 25.9, 49.8, 72.6, 93.3, 149, 192, 301 mg B/L
Measured concentrations for test with Hardness 200 : 0.05, 0.1, 0.49, 1.04, 4.6, 6.23, 8.82, 29.5, 49.1, 103, 197, 320 mg B/L

Details on test conditions:

TEST SYSTEM
Fish and amphibian eggs were cultured through 4 days posthatching in Pyrex chambers, through which test water was perfused at prescribed flow rates. The toxicant was administered to a mixing chamber situated ahead of each culture dish, using graduated flow from a syringe pump. Synthetic culture water was delivered to the mixing chamber by regulated flow from a peristaltic pump. Flow rates from both syringe and peristaltic pumps were monitored by liquid flow meters. Flow rate was set at 200 ml /hr for 300 ml test chambers. Syringe and peristaltic pumps were provided with calibrated, continuously variable speed controls. The concentration of toxicant delivered to the mixing chamber was regulated by adjusting the mixing ratio from the pumping units and/or by varying the concentration of toxicant delivered from the syringe pump. Solutions from the two channels were mixed with a Teflon-coated magnetic stirring bar, and delivered to the culture chamber under positive pressure.
The system was operated using Brinkmann (model 131900) and Gilson (model HP8) multichannel peristaltic pumps and Sage syringe pumps (model 355)). The latter was fitted with a modified syringe holder, and operated using 10 ml double-ground, glass syringes. Syringes were selected for equal stroke volume, and peristaltic pump channels were fitted with tubing of matched diameters.
For test concentrations of 10 ppm or more, boron compounds generally were added directly to the culture water in the peristaltic pump reservoir, eliminating the need for the syringe pump channel. In such instances, the toxicants remained stable at selected test concentrations for 24 hours or more in polyethylene containers, and important test parameters of culture water were not altered upon standing.
All aquatic bioassay cultures were maintained in walk-in environmental rooms. Culture water was given continuous aeration in the peristaltic pump reservoirs.



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Culture water was prepared from distilled, double deionized water, having a conductivity of 0.25 umhos or less. Routine monitoring was conducted for background contaminants, including mercury, cadmium, lead and other trace metals. Reagent grade calcium, magnesium, sodium and potassium salts were added. The basic stock was prepared to give a water hardness level of 200 ppm (as CaCO3) and a pH of 7.5 - 8.0. Different levels of hardness were achieved by dilution of the basic culture water.
- Chlorine: 98.2 mg/L
- Alkalinity: 82.0 mg/L
- Conductivity: 300 umhos
- Intervals of water quality measurement: daily intervals


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

Duration:

9 d

Dose descriptor:

LC10

Effect conc.:

47 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Remarks:

+ teratogenicity

Remarks on result:

other: Hardness 50; confidence limits: 18.1 - 122.0 mg/L

Duration:

9 d

Dose descriptor:

LC10

Effect conc.:

17.3 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Remarks:

+ teratogenicity

Remarks on result:

other: Hardness 200; confidence limits: 10.8 - 27.8 mg/L

Reported statistics and error estimates:

For Birge & Black : Log probit analysis was used to statistically determine LC01 and LC50 values. Analysis of variance and the t-test were used to determine statistical significance of differences between boric acid and borax toxicity, water hardness levels and other test variables.
The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A.

Reference 6

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Peer-reviewed technical publication. Duration of early-lifestage test shorter than current method

Qualifier:

no guideline followed

Principles of method if other than guideline:

Embryos were exposed to different concentrations of boric acid starting at fertilization and continuing through 8 days posthatching.

GLP compliance:

not specified

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Micropterus salmoides

Details on test organisms:

TEST ORGANISM
- Common name: Largemouth bass
- Source: Frankfort National Fish hatchery, Frankfort, Kentucky
- Exposure of embryos was initiated within 2 to 4 hours after spawning and maintained through 8 days posthatching. Hatching time averaged 3 days


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: Fertilized eggs were transported in hatchery water at ambient temperature, using a portable cooler.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

11 d

Hardness:

204 mg/L CaCO3

Test temperature:

20 °C

pH:

7.5

Dissolved oxygen:

8.4 mg/L

Nominal and measured concentrations:

Nominal: 0.01, 0.1, 1.0, 10.0, 25.0, 50.0 mg/L
Measured: 0.009, 0.109, 1.39, 12.17, 25.08, 51.13 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): temperature-regulated environmental chamber using a flow-through system
- Type of flow-through (e.g. peristaltic or proportional diluter): Birge et al (1981)
- No. of fertilized eggs/embryos per vessel: > 80
- No. of vessels per concentration (replicates): 2 - 4


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The reconstitute water was prepared by the addition of reagent grade calcium, magnesium, sodium, and potassium salts to distilled, deionized water. The water was formulated to have a hardness of 200 mg/L as CaCO3. A full description of the chemical constituents has been described earlier (Birge and Cassidy 1983)
- Conductivity: 229µmhos/cm
- Intervals of water quality measurement: 1 to 2 day intervals


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Embryos and larvae were examined daily to gauge extent of development and to remove dead specimens.

Duration:

32 d

Dose descriptor:

LC10

Effect conc.:

16.8 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Remarks on result:

other: confidence limits: 14.8-19.0 mg/L

Details on results:

The authors reported NOEC value of 1.39 mg/L which is not reliable because of dilution factor of 10; LC10 is reliable but calculated from an uncomplete dose-response. Indeed, only 6 Boron concentrations are reported by Black et al (1993). The results of the same experiment have been reported by Birge and Black (1981) but with a more comprehensive dose reponse curve, with 11 Boron concentrations, resulting in a more reliable LC10 of 36.8 mg/L.

Reported statistics and error estimates:

Statistical differences were analyzed using the approach by Freund & Littell (1981). The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A.

Reference 7

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well performed study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Test animals were exposed to 10 - 14 concentrations of boric acid and borax at 2 different water hardness levels.

GLP compliance:

not specified

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Carassius auratus

Details on test organisms:

TEST ORGANISM
- Common name: Goldfish
- Source: Gravid fish and amphibians were obtained from State and Federal hatcheries at Frankfort, Kentucky, Erwin, Tennessee, and Senecaville, Ohio, and from selected sites within Kentucky.


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: collected from natural spawn

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Hardness:

Hardness 50 : 46.2 mg/L
Hardness 200 : 194.8 mg/L

Test temperature:

24.8 - 27°C

pH:

Hardness 50 : 8.3
Hardness 200 : 8.1

Dissolved oxygen:

ca. 10 mg/L

Nominal and measured concentrations:

Nominal concentrations : 0.05, 0.10, 0.50, 1.00, 5.00, 7.50, 10.00, 25.00, 50.00, 100.00, 200.00, 300.00 mg B/L
Measured concentrations for test with Hardness 50 : 0.05, 0.07, 0.54, 0.94, 4.68, 7.43, 9.1, 26.5, 48.75, 96, 189.67, 327.6 mg B/L
Measured concentrations for test with Hardness 200 : 0.05, 0.08, 0.69, 0.89, 4.1, 6.47, 8.53, 27.33, 42.25, 88.67, 190, 303 mg B/L

Details on test conditions:

TEST SYSTEM
Fish and amphibian eggs were cultured through 4 days posthatching in Pyrex chambers, through which test water was perfused at prescribed flow rates. The toxicant was administered to a mixing chamber situated ahead of each culture dish, using graduated flow from a syringe pump. Synthetic culture water was delivered to the mixing chamber by regulated flow from a peristaltic pump. Flow rates from both syringe and peristaltic pumps were monitored by liquid flow meters. Flow rate was set at 200 ml /hr for 300 ml test chambers. Syringe and peristaltic pumps were provided with calibrated, continuously variable speed controls. The concentration of toxicant delivered to the mixing chamber was regulated by adjusting the mixing ratio from the pumping units and/or by varying the concentration of toxicant delivered from the syringe pump. Solutions from the two channels were mixed with a Teflon-coated magnetic stirring bar, and delivered to the culture chamber under positive pressure.
The system was operated using Brinkmann (model 131900) and Gilson (model HP8) multichannel peristaltic pumps and Sage syringe pumps (model 355)). The latter was fitted with a modified syringe holder, and operated using 10 ml double-ground, glass syringes. Syringes were selected for equal stroke volume, and peristaltic pump channels were fitted with tubing of matched diameters.
For test concentrations of 10 ppm or more, boron compounds generally were added directly to the culture water in the peristaltic pump reservoir, eliminating the need for the syringe pump channel. In such instances, the toxicants remained stable at selected test concentrations for 24 hours or more in polyethylene containers, and important test parameters of culture water were not altered upon standing.
All aquatic bioassay cultures were maintained in walk-in environmental rooms. Culture water was given continuous aeration in the peristaltic pump reservoirs.



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Culture water was prepared from distilled, double deionized water, having a conductivity of 0.25 umhos or less. Routine monitoring was conducted for background contaminants, including mercury, cadmium, lead and other trace metals. Reagent grade calcium, magnesium, sodium and potassium salts were added. The basic stock was prepared to give a water hardness level of 200 ppm (as CaCO3) and a pH of 7.5 - 8.0. Different levels of hardness were achieved by dilution of the basic culture water.
- Chlorine: 98.2 mg/L
- Alkalinity: 82.0 mg/L
- Conductivity: 300 umhos
- Intervals of water quality measurement: daily intervals


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

Duration:

7 d

Dose descriptor:

LC10

Effect conc.:

37.8 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Remarks:

+ teratogenicity

Remarks on result:

other: Hardness 50; confidence limits: 31.0 - 46.1 mg/L

Duration:

7 d

Dose descriptor:

LC10

Effect conc.:

37.8 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Remarks:

+ teratogenicity

Remarks on result:

other: Hardness 200; confidence limits: 33.6 - 42.6 mg/L

Reported statistics and error estimates:

For Birge & Black : Log probit analysis was used to statistically determine LC01 and LC50 values. Analysis of variance and the t-test were used to determine statistical significance of differences between boric acid and borax toxicity, water hardness levels and other test variables.
The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A.

Reference 8

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: ASTM E1241-05 Standard Guide for Conducting Early Life-Stage Toxicity Tests with Fishes

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

GLP compliance:

no

Remarks:

study is not in compliance with GLP

Analytical monitoring:

yes

Details on sampling:

Each treatment sampled at beginning and end of test

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Source: Aquatic Biosystems, Fort Collins Colorado

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): done by supplier
- Method of collection of fertilised eggs: done by supplier
- Subsequent handling of eggs: Shipped via express delivery. Test begun upon arrival

POST-HATCH FEEDING
- Type/source of feed: brine shrimp
- Amount given:
- Frequency of feeding: 2x per day - days 0-2 and 5 days per week for rest of test. 1x per day on weekends (2 days per week)

Test type:

semi-static

Water media type:

freshwater

Total exposure duration:

32 d

Hardness:

84 to 94

Test temperature:

23.6 to 25.5

pH:

7.5 to 8..2

Dissolved oxygen:

5.13 to 7.5

Nominal and measured concentrations:

Nominal: control, 2.75, 5.5, 11, 22, 44 mg-B/L
Measured: 0.03, 2.8, 5.7, 11.2, 23, 44.5 mg-B/L

Details on test conditions:

TEST SYSTEM
- Test vessel: day 0-2 1-L beakers, days 2-32 600 ml beakers
- Type : open
- Material, size, headspace, fill volume: glass, day 0-2 1L, days 2-32 500 ml
- Aeration: days 0-2 only
- Renewal rate of test solution: 90% every 3 days

- No. of fertilized eggs/embryos per vessel: day 0-2 60/chamber, days 2-32 10/chamber
- No. of vessels per concentration (replicates): day 0-2 one per concentration, days 2-32 four per concentration
- No. of vessels per control (replicates): day 0-2 one per concentration, days 2-32 four per concentration

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: MHRW (moderately hard reconstituted water) per US EPA 2002
- Total organic carbon: 0 nominal
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine: 0 nominal
- Ca/mg ratio: 1.46
- Conductivity:
- Salinity:
- Culture medium different from test medium: Aquatic Biosystems Lab water, hardness 114 mg/L, alkalinity 115 mg/L
- Intervals of water quality measurement: boron, pH, conductivity, alkalinity, hardness on "in" and "out" water on every changeover, temperature and D.O. daily

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16 L: 8 D

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Survival (daily), dry weight per 10 fish (at conclusion of test)


RANGE-FINDING STUDY

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

11.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

mortality

Duration:

32 d

Dose descriptor:

LOEC

Effect conc.:

23 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

mortality

Duration:

32 d

Dose descriptor:

other: MATC

Effect conc.:

16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

mortality

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

>= 44.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

weight

Remarks on result:

other: (dry weight per 10 fish)

Duration:

32 d

Dose descriptor:

LC10

Effect conc.:

21.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

mortality

Remarks on result:

other: confidence limits: 19.3-24.2 mg/L

Reported statistics and error estimates:

Data tested for normality using the Shapiro-Wilk's test, and for homogeneity of variance using Bartlett's test. Data passing both tests were analyzed for differences between group and control using Dunnett's test. Those not passing both tests were analysed using Steele's Many-One test.
The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A

Survival Data

Day	Control	2.8	5.7	11.2	23	44.5
3	100.0%	100.0%	100.0%	100.0%	100.0%	100.0%
7	100.0%	97.5%	95.0%	97.5%	95.0%	97.5%
14	100.0%	92.5%	87.5%	95.0%	92.5%	85.0%
21	97.5%	92.5%	87.5%	95.0%	87.5%	42.5%
28	95.0%	92.5%	87.5%	95.0%	80.0%	20.0%
32	95.0%	92.5%	87.5%	95.0%	80.0%	15.0%

Dry weight data (mg)

Day	Control	2.8	5.7	11.2	23	44.5
32	0.8926	0.9336	0.7946	0.8595	n/a	n/a

Reference 9

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well performed study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Test animals were exposed to 10 - 14 concentrations of boric acid and borax at 2 different water hardness levels.

GLP compliance:

no

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: Gravid fish and amphibians were obtained from State and Federal hatcheries at Frankfort, Kentucky, Erwin, Tennessee, and Senecaville, Ohio, and from selected sites within Kentucky.


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: Trout eggs and sperm were collected for test purposes by artificial spawning and milking, using methods of Leitritz. Fertilization was accomplished by mixing sperm and eggs for 15 minutes immediately prior to the onset of boron exposure.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Hardness:

Hardness 50 : 49 mg/L
Hardness 200 : 191 mg/L

Test temperature:

13 - 14°C

pH:

Hardness 50 : 7.9
Hardness 200 : 7.8

Dissolved oxygen:

ca. 10 mg/L

Nominal and measured concentrations:

Nominal concentrations : 0.05, 0.10, 0.50, 1.00, 5.00, 7.50, 10.00, 25.00, 50.00, 100.00, 200.00, 300.00 mg B/L
Measured concentrations for test with Hardness 50 : 0.001, 0.01, 0.12, 0.49, 0.96, 9.7, 22.6, 50.6, 97.4, 190.2 mg B/L
Measured concentrations for test with Hardness 200 : 0.001, 0.01, 0.1, 0.5, 0.94, 4.98, 9.63, 49.7, 100, 191 mg B/L

Details on test conditions:

TEST SYSTEM
Fish and amphibian eggs were cultured through 4 days posthatching in Pyrex chambers, through which test water was perfused at prescribed flow rates. The toxicant was administered to a mixing chamber situated ahead of each culture dish, using graduated flow from a syringe pump. Synthetic culture water was delivered to the mixing chamber by regulated flow from a peristaltic pump. Flow rates from both syringe and peristaltic pumps were monitored by liquid flow meters. Flow rate was set at 200 ml /hr for 300 ml test chambers. Syringe and peristaltic pumps were provided with calibrated, continuously variable speed controls. The concentration of toxicant delivered to the mixing chamber was regulated by adjusting the mixing ratio from the pumping units and/or by varying the concentration of toxicant delivered from the syringe pump. Solutions from the two channels were mixed with a Teflon-coated magnetic stirring bar, and delivered to the culture chamber under positive pressure.
The system was operated using Brinkmann (model 131900) and Gilson (model HP8) multichannel peristaltic pumps and Sage syringe pumps (model 355)). The latter was fitted with a modified syringe holder, and operated using 10 ml double-ground, glass syringes. Syringes were selected for equal stroke volume, and peristaltic pump channels were fitted with tubing of matched diameters.
For test concentrations of 10 ppm or more, boron compounds generally were added directly to the culture water in the peristaltic pump reservoir, eliminating the need for the syringe pump channel. In such instances, the toxicants remained stable at selected test concentrations for 24 hours or more in polyethylene containers, and important test parameters of culture water were not altered upon standing.
All aquatic bioassay cultures were maintained in walk-in environmental rooms. Culture water was given continuous aeration in the peristaltic pump reservoirs.



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Culture water was prepared from distilled, double deionized water, having a conductivity of 0.25 umhos or less. Routine monitoring was conducted for background contaminants, including mercury, cadmium, lead and other trace metals. Reagent grade calcium, magnesium, sodium and potassium salts were added. The basic stock was prepared to give a water hardness level of 200 ppm (as CaCO3) and a pH of 7.5 - 8.0. Different levels of hardness were achieved by dilution of the basic culture water.
- Chlorine: 98.2 mg/L
- Alkalinity: 82.0 mg/L
- Conductivity: 300 umhos
- Intervals of water quality measurement: daily intervals


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

Duration:

28 d

Dose descriptor:

LC10

Effect conc.:

9.9 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Remarks:

+ teratogenicity

Remarks on result:

other: Hardness 50; confidence limits: 7.7 - 12.9 mg/L

Duration:

28 d

Dose descriptor:

LC10

Effect conc.:

40.2 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Remarks:

+ teratogenicity

Remarks on result:

other: Hardness 200; confidence limits: 32.3 - 50.0 mg/L

Reported statistics and error estimates:

Log probit analysis was used by the authors to statistically determine LC01 and LC50 values. The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A.

Reference 10

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well performed study

Principles of method if other than guideline:

no guideline was reported

GLP compliance:

no

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: Gravid fish and amphibians were obtained from State and Federal hatcheries at Frankfort, Kentucky, Erwin, Tennessee
- Boron treatment was initiated subsequent to fertilization


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: Trout eggs and sperm were collected for test purposes by artificial spawning and milking, using methods of Leitritz. Fertilization was accomplished by mixing sperm and eggs for 15 minutes immediately prior to the onset of boron exposure.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Hardness:

197 mg/L

Test temperature:

13.2 °C

pH:

7.4

Dissolved oxygen:

9.8 mg/L

Nominal and measured concentrations:

Nominal concentrations : 0.01, 0.10, 1.00, 10.00, 25.0, 50.0, 100.00, 200.00, 300.00, 400.0, 500.0 mg B/L
Measured concentrations: 0.008, 0.1, 1.26, 10.19, 24.98, 49.36, 100.0, 196.1, 383.3, 482.2 mg B/L

Details on test conditions:

TEST SYSTEM
Fish eggs were cultured through 4 days posthatching in Pyrex chambers, through which test water was perfused at prescribed flow rates. The toxicant was administered to a mixing chamber situated ahead of each culture dish, using graduated flow from a syringe pump. Synthetic culture water was delivered to the mixing chamber by regulated flow from a peristaltic pump. Flow rates from both syringe and peristaltic pumps were monitored by liquid flow meters. Flow rate was set at 200 ml /hr for 300 ml test chambers. Syringe and peristaltic pumps were provided with calibrated, continuously variable speed controls. The concentration of toxicant delivered to the mixing chamber was regulated by adjusting the mixing ratio from the pumping units and/or by varying the concentration of toxicant delivered from the syringe pump. Solutions from the two channels were mixed with a Teflon-coated magnetic stirring bar, and delivered to the culture chamber under positive pressure.
The system was operated using Brinkmann (model 131900) and Gilson (model HP8) multichannel peristaltic pumps and Sage syringe pumps (model 355)). The latter was fitted with a modified syringe holder, and operated using 10 ml double-ground, glass syringes. Syringes were selected for equal stroke volume, and peristaltic pump channels were fitted with tubing of matched diameters.
For test concentrations of 10 ppm or more, boron compounds generally were added directly to the culture water in the peristaltic pump reservoir, eliminating the need for the syringe pump channel. In such instances, the toxicants remained stable at selected test concentrations for 24 hours or more in polyethylene containers, and important test parameters of culture water were not altered upon standing.
All aquatic bioassay cultures were maintained in walk-in environmental rooms. Culture water was given continuous aeration in the peristaltic pump reservoirs.



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Culture water was prepared from distilled, double deionized water, having a conductivity of 0.25 umhos or less. Routine monitoring was conducted for background contaminants, including mercury, cadmium, lead and other trace metals. Reagent grade calcium, magnesium, sodium and potassium salts were added. The basic stock was prepared to give a water hardness level of 200 ppm (as CaCO3) and a pH of 7.5 - 8.0. Different levels of hardness were achieved by dilution of the basic culture water.
- Alkalinity: 65 mg/L
- Conductivity: 282 umhos
- Intervals of water quality measurement: daily intervals


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

Duration:

28 d

Dose descriptor:

LC10

Effect conc.:

5.1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Remarks:

+ teratogenicity

Remarks on result:

other: confidence limits: 2.4 - 10.7 mg/L

Duration:

28 d

Dose descriptor:

LC10

Effect conc.:

31.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Remarks on result:

other: confidence limits: 10.0 - 98.5 mg/L

Reported statistics and error estimates:

Log probit analysis was used to statistically determine LC01 and LC50 values. The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A

Reference 11

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Well performed study for the test at 50 mg/L hardness. The test result from the hardness 200 mg/L experiment revealed a LC10 value of 0.15 mg/L. However, this value is unrelaible because of the high uncertainty/confidence limits between 0.0008 - 25.4 mg/L, and of the unclear and partial dose-response curve. Indeed, in the highest concentration revealed an maximum effect on mortality of 50%.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Test animals were exposed to 10 - 14 concentrations of boric acid at 2 different water hardness levels.

GLP compliance:

no

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: Gravid fish and amphibians were obtained from State and Federal hatcheries at Frankfort, Kentucky, Erwin, Tennessee, and Senecaville, Ohio, and from selected sites within Kentucky.
- Boron treatment was initiated subsequent to fertilization


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: Trout eggs and sperm were collected for test purposes by artificial spawning and milking, using methods of Leitritz. Fertilization was accomplished by mixing sperm and eggs for 15 minutes immediately prior to the onset of boron exposure.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Hardness:

Hardness 50 : 54.1 mg/L
Hardness 200 : 204 mg/L

Test temperature:

13 - 14 °C

pH:

Hardness 50 : 7.7
Hardness 200 : 7.9

Dissolved oxygen:

ca. 10 mg/L

Nominal and measured concentrations:

Nominal concentrations : 0.05, 0.10, 0.50, 1.00, 5.00, 7.50, 10.00, 25.00, 50.00, 100.00, 200.00, 300.00 mg B/L
Measured concentrations for test with Hardness 50 : 0.001, 0.01, 0.11, 1, 4.74, 9.26, 23.5, 45.5, 94, 190 mg B/L
Measured concentrations for test with Hardness 200: 0.001, 0.01, 0.1, 0.47, 0.98, 4.85, 9.4, 23.8, 48.3, 100.2, 186 mg B/L

Details on test conditions:

TEST SYSTEM
Fish and amphibian eggs were cultured through 4 days posthatching in Pyrex chambers, through which test water was perfused at prescribed flow rates. The toxicant was administered to a mixing chamber situated ahead of each culture dish, using graduated flow from a syringe pump. Synthetic culture water was delivered to the mixing chamber by regulated flow from a peristaltic pump. Flow rates from both syringe and peristaltic pumps were monitored by liquid flow meters. Flow rate was set at 200 ml /hr for 300 ml test chambers. Syringe and peristaltic pumps were provided with calibrated, continuously variable speed controls. The concentration of toxicant delivered to the mixing chamber was regulated by adjusting the mixing ratio from the pumping units and/or by varying the concentration of toxicant delivered from the syringe pump. Solutions from the two channels were mixed with a Teflon-coated magnetic stirring bar, and delivered to the culture chamber under positive pressure.
The system was operated using Brinkmann (model 131900) and Gilson (model HP8) multichannel peristaltic pumps and Sage syringe pumps (model 355)). The latter was fitted with a modified syringe holder, and operated using 10 ml double-ground, glass syringes. Syringes were selected for equal stroke volume, and peristaltic pump channels were fitted with tubing of matched diameters.
For test concentrations of 10 ppm or more, boron compounds generally were added directly to the culture water in the peristaltic pump reservoir, eliminating the need for the syringe pump channel. In such instances, the toxicants remained stable at selected test concentrations for 24 hours or more in polyethylene containers, and important test parameters of culture water were not altered upon standing.
All aquatic bioassay cultures were maintained in walk-in environmental rooms. Culture water was given continuous aeration in the peristaltic pump reservoirs.



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Culture water was prepared from distilled, double deionized water, having a conductivity of 0.25 umhos or less. Routine monitoring was conducted for background contaminants, including mercury, cadmium, lead and other trace metals. Reagent grade calcium, magnesium, sodium and potassium salts were added. The basic stock was prepared to give a water hardness level of 200 ppm (as CaCO3) and a pH of 7.5 - 8.0. Different levels of hardness were achieved by dilution of the basic culture water.
- Chlorine: 98.2 mg/L
- Alkalinity: 82.0 mg/L
- Conductivity: 300 umhos
- Intervals of water quality measurement: daily intervals


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

Duration:

28 d

Dose descriptor:

LC10

Effect conc.:

41.5 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Basis for effect:

mortality

Remarks:

+ teratogenicity

Remarks on result:

other: Hardness 50; confidence limits: 21.0 - 82.1 mg/L

Details on results:

The test result from the Hardness 200 mg/L experiment revealed a LC10 value of 0.15 mg/L. However, this value is unrelaible because of the high uncertainty/confidence limits between 0.0008 - 25.4 mg/L, and of the unclear and partial dose-response curve. Indeed, in the highest concentration revealed an maximum effect on mortality of 50%.

Reported statistics and error estimates:

Log probit analysis was used by the authors to statistically determine LC01 and LC50 values. The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A.

Description of key information

Long-term effects (LC₁₀) on freshwater fish ranged from 3.5 to 47 mg B/L. Adequate long-term LC₁₀ of 21.6 mg B/L was found for the fresh water fish P. promelas in a study according to EPA OPPTS 850.1400 (Soucek, 2010).