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Long-term toxicity to aquatic invertebrates

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long-term toxicity to aquatic invertebrates
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 211 (Daphnia magna Reproduction Test)
according to
other: EU New Draft Guideline "Prolonged toxicity study with Daphnia magna (inhibition of reproduction)"
GLP compliance:

Test material

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:
Details on sampling:
The samples of approximately 50 ml were received in polyethylene bottles from the Department of Environmental Toxicology of the TNO Nutrition and Food Research Institute on 5 August and 20 October 1999. Upon receipt they were identified with the unique codes. They were analysed on 11 August and 22 November 1999.

Test solutions


Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Source: fresh-water crustacean Daphnia magna, cultrued in the laboratory under standard conditions. Daphnia magna has been cultured at TNO in the dilution water used for the present test
- Feeding during test
- Food type: cells of the unicellular alga Chlorella sp. and some 'yeast' was added as extra food
- Frequency: daily

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
21 d

Test conditions

212 mg/L (as CaCO3)
Test temperature:
19.3 - 20.9 °C
7.2 - 8.0
Dissolved oxygen:
> 7.4 mg/L
Nominal and measured concentrations:
Nominal concentration : 1.8, 3.2, 5.6, 10, 18, 32 and 56 mg B/L.
Measured concentrations: 0.57, 2.59, 3.92, 6.55, 10.78, 19.49, 34.17 and 59.14 mg/L
The actual measured boron concentrations were between 98% and 102% of the nominal values in the newly prepared test solutions and between 105 and 108% in the spent solutions
Details on test conditions:
- Test vessel:
- Material, size, headspace, fill volume: 100 ml all-glass beakers, each containing 50 ml of exposure medium.
- Aeration: no aeration
- Renewal rate of test solution (frequency/flow rate): every Monday, Wednesday and Friday
- No. of organisms per vessel: 1

- Source/preparation of dilution water: DSWL-E prepared from ground water
- 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 and oxygen concentrations were measured weekly in one of the newly prepared control test vessels and in one vessel of each test substance concentration. The pH and oxygen concentration were also measured in the same spent solutions, just before replacement and in any test vessel with a dead animal. The pH of the exposure media was not adjusted to that of the dilution water. The temperature was measured in one of the control vessels and the highest concentration tested just before and just after replacement of the test media.

- Photoperiod: 16 light - 8 dark with transition periods of 30 min

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The dead animals were recorded daily, and removed if applicable, and any young born were counted and removed at each replacement time and at the end of the test. The condition (swimming behaviour, colour or any other visually observable morphological or behavioural criterion) and the size of the original test animals were qualitatively compared with those of the control animals at the same time.

- Appropriate concentrations for the toxicity tests were determined in a preliminary range finding test.

Results and discussion

Effect concentrationsopen allclose all
21 d
Dose descriptor:
Effect conc.:
10.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
Basis for effect:
21 d
Dose descriptor:
Effect conc.:
17.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
Basis for effect:
Remarks on result:
other: confidence limits: 15.5 - 20.2 mg/L
21 d
Dose descriptor:
Effect conc.:
34.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
Basis for effect:
Details on results:
NOEC and LC10 values have been calculated for the endpoint reproduction. Preference is given to the use of the EC10 (17.7 mg/L) compared to the NOEC (10.8 mg/L ) value for reporduction. For the endpoint mortality, both the NOEC (34.2 mg/L) and LC10 (44.5 mg/L) values could be calculated. However the LC10 for the endpoint mortality was not reliable because of the large confidence interval between 0 mg/L - infinity.
Reported statistics and error estimates:
Statistical significance for mortality was determined with a binomial test at a 95 % and 99 % significance level. Statistical significance for reproduction was dermined using the two-tailed Dunnett-test at a 95 % and 99 % significance level using the mean number of young per female as observed values. In both cases, the obersvations at each concentrion were compared with those of the control. In the case of significance at the 99% level only that significance is given.

The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A

Any other information on results incl. tables

If the reproduction of Daphnia magna is inhibited at sublethal concentrations the (cumulative) reproduction can often be described by: Ri(c) = R(0)/[1 + (c/EC50)^b] + ei in which Ri is the (cumulative) reproduction in the ith test vessel. R depends on the concentration of the test compound c. EC50 is the concentration at which the reproduction is reduced to 50% of the reproduction in the unstressed control. b is a slope parameter ( the larger b, the steeper the slope of the relation between concentration and reproduction). In each experiment a certain error, ei, will occur. These errors are assumed to be independent and normal distributed with expectation 0 and variance σ². Given these assumptions about the relation between concentration and reproduction and about the error in the observations the maximum likelihood estimations of the parameters of the function are calculated. Estimation of the LC50 and its confidence interval : At a given time, the mortality probability of an individual is assumed to be logistically related to the logarithm of the concentration, i.e. pi = (ei+p0)/(1+ei), where ei = (ci/α)^(1/β) and pi is the mortality probability in the ith concentration p0 is the mortality probability in concentration α is the LC50 β is a parameter inversely proportional to the maximum gradient of the dose response function ci is the ith concentration The parameters p0, α and β are estimated from the counts by means of the maximum likelihood method; i.e. the parameter values to be selected maximize the probablity of the counts as a function of the three parameters. Since the distribution of α will not be symmetrical the variance-covariance matrix is not estimated for the parameters p0, α and β themselves, but for p0, γ = ln α and β. The variance-covariance matrix is estimated by the inverse of the information matrix. The 95% confidence interval of the LC50 is now given as. α.exp(+-2[var(γ)]^(1/2))= α.exp(+-2[var(ln(α))]^(1/2))

Duration Endpoint Effect conc. Nominal/Measured Conc. based on Basis for effect Remarks (e.g. 95% CL)
21 d EC50 22 mg/L nominal element reproduction
21 d LOEC 18 mg/L nominal element reproduction
21 d LC50 34 mg/L nominal element mortality
21 d LOEC 56 mg/L nominal element mortality
 21 d LOEC  32 mg/L nominal  element  condition  

Applicant's summary and conclusion