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Ecotoxicological information

Long-term toxicity to fish

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Administrative data

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

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2000
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010
Reference Type:
review article or handbook
Title:
Unnamed
Year:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Boric Acid, Manufacturing Grade
- Molecular formula (if other than submission substance): H3BO3
- Physical state: white powder
- Analytical purity: +99.9%
- Lot/batch No.: not given
- Storage condition of test material: stored in one litre square polyethylene bottle at room temperature

Sampling and analysis

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.

Test solutions

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

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.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
34 d

Test conditions

Hardness:
212 mg
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.

Results and discussion

Effect concentrationsopen allclose all
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
5.6 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
mortality
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
5.6 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
length
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
5.6 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
weight
Remarks on result:
other: (see discussion of statistical analysis below)
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
5.6 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: condition
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. The study authors excluded the mean dry weight data from Dose 1 from the Dunnett's analysis, but included data from that group in all other analyses. The NOEC for condition was not determined statistically.

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 the study authors.

Any other information on results incl. tables

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

Applicant's summary and conclusion