Humic acids and fulvic acids extracted from leonardite, reaction product with formaldehyde, potassium hydrogen sulfite, sodium hydrogen sulfite and potassium hydroxide

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

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

Adequacy of study:

key study

Study period:

from Sempember 24, 2007 to November 29, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was carried out in accordance with internationally valid GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Humic acids, potassium salts

EC Number:

271-030-1

EC Name:

Humic acids, potassium salts

Cas Number:

68514-28-3

IUPAC Name:

68514-28-3

Details on test material:

- Name of test material (as cited in study report): Humic acids, potassium salts
- Molecular formula: not known - UVCB substance
- Molecular weight: not known - UVCB substance
- Substance type: technical product
- Physical state: solid
- Lot/batch No.: 16. 5. 2007/R
- Expiration date of the lot/batch: 05/2022
- Stability under test conditions: stable
- Storage condition of test material: dry conditions

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 61 - 150 mg/L (nominal concentration)
- Sampling method:
The analytical determination of the test substance concentrations was performed in each test concentrations at the beginning and at the end of the test.
- Sample storage conditions before analysis:
The samples were analysed on the same day as sampling.
All samples were stored at laboratory temperature.

Test solutions

Vehicle:

no

Details on test solutions:

The test substance was soluble in the dilution water in concentration needed for the test. The stock solution of the test substance was prepared in the dilution water. 200 mg of the test substance was weighed into 2000 mL of the dilution water for preliminary test and 2000 mg of the test substance was weighed into 2000 mL of the dilution water for limit test. The concentrations of solutions used in the preliminary and the limit test were obtained by dilution of the stock solution with dilution water. The exact concentrations were analyzed.

Test organisms

Test organisms (species):

Poecilia reticulata

Details on test organisms:

TEST ORGANISM
- Common name: Poecilia reticulata
- Strain:
- Source: certified supplier BioTest s.r.o., Konárovice (Czech Republic).
- Age at study initiation (mean and range, SD): 3-4months old
- Length at study initiation (length definition, mean, range and SD): 2.0-3.0 cm
- Weight at study initiation (mean and range, SD): preliminary test 7 fishes cca 1g, limit test: 10 fishes cca 0.7 g
- Method of breeding: The fish were kept in all-glass aquaria
- Feeding during test: no
- Food type:
- Amount:
- Frequency:


ACCLIMATION
- Acclimation period: Fish were acclimatized for 7 days
- Acclimation conditions (same as test or not): in dilution water
- Type and amount of food: Diet: “Tropica” - feed for aquarium fish, ad libitum and sometimes live daphnia. The fishes were not fed during 72 hours before the test.
- Feeding frequency: ad libitum
- Health during acclimation (any mortality observed): healthy animals

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

none

Test conditions

Hardness:

Content of (Ca + Mg) ions: 2.63 mmol/L (preliminary test)
Content of (CA + Mg) ions: 2.59 mmol/L (main test)

Test temperature:

22±2°C, with a variation of ± 1°C at each individual test

pH:

7.8 - 7.9 (at the beginning)

Dissolved oxygen:

8.5 mg/L at temperature 21.2 °C (main test)

Nominal and measured concentrations:

see table 1

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: open
- Material, size, headspace, fill volume: Glass vessels, size 3L, volume 2L
- Aeration: without
- No. of organisms per vessel: 7 for the preliminary test, 10 for the limit test
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Standard dilution water was prepared just before the test by admixing 2.5 mL of stored solutions A, B, C and D in 1000 mL of distilled water.
A. 117.6 g CaCl2.2H20 in 1L distilled water
B. 49.3 g MgSO4.7H2O in 1L distilled water
C. 25.9 g NaHCO3 in 1L distilled water
D. 2.3 g KCl in 1L distilled water
The dilution water should be of pH 7.8 ± 0.2. It is saturated by oxygen by the aeration at temperature of 20 - 24°C. The content of sum Ca + Mg ions should have the value of 2.5 mmol.L-1.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: daily
- Light intensity: daylight

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 128 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 2

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 128 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

> 128 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 4

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 128 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 5

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

128 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 6

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

128 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 7

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

128 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 8

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

128 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 9

Duration:

24 h

Dose descriptor:

LC100

Effect conc.:

> 128 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 10

Duration:

48 h

Dose descriptor:

LC100

Effect conc.:

> 128 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 11

Duration:

72 h

Dose descriptor:

LC100

Effect conc.:

> 128 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 12

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 128 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: no
- Mortality of control: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
The analytical results showed, that the composition of the dilution water has influence on the solubility of the test substance and the part of the test substance was precipitated at preparation of the stock solution. For this reason concentrations measured at the beginning of the test are lower than nominal concentrations.

Results with reference substance (positive control):

Reference test
The sensitivity of the test species and correctness of test performance is periodically verified in three-month period by testing the reference substance, potassium dichromate.
The results of verification test with K2Cr2O7, carried out in period from 17.9. to 21.9.2007, are the following:

48h – LC50 = 141 mg.L-1 (95 % confidence limit: 133 – 148 mg.L-1)
96h – LC50 = 133 mg.L-1 (95 % confidence limit: 131 – 135 mg.L-1)

The results of the last interlaboratory test arranged by Centre for Assessment of Laboratories of T.G.Masaryk Water Research Institute Prague in 2006 for potassium dichromate were:

96 h - LC50 = 96 – 154 mg.L-1

The mortality of test organisms in the reference substance solution meets criteria determined by the interlaboratory test.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Validity of the test

Test quality criteria

These quality criteria are given in the guideline.

The result of test should satisfy quality criteria as follows:

1. Mortality in the control must not exceed 10 % (or one fish if less than ten are used) by the end of the test.
2. The dissolved oxygen concentration must be at least 60 % of initial value.
3. The measured values of pH solution should not change more than 1 pH unit.
4. The test substance concentration in test mixtures should not decrease under 80 % of the initial concentration.

Quality criteria at points 1, 2, 3 and 4 were fulfilled.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Executive summary:

The test substance was tested for acute toxicity on fish Poecilia reticulata.

The test was performed according to EU Method C.1., Acute toxicity for fish, Directive 92/69/EEC. Published in O.J. L 383A, 1992.

The water solubility of the test substance is 279 mg·L^-1 (see Study No. 26/07/46: Humic acids, potassium salts - Water solubility).

The preliminary test was performed in range of test substance nominal concentrations 1 – 100 mg·L^-1. The analytical results showed, that the composition of the dilution water has influence on the solubility of the test substance and the part of the test substance is precipitated at preparation of the stock solution. For this reason concentrations measured at the beginning of the test are lower than nominal concentrations. Based on no toxicity of the test substance found in preliminary test, the limit test was performed.

The limit test was performed in range of test substance nominal concentrations 61 – 150 mg·L^-1. The test was performed as static.

With regards to analytical results obtained by preliminary test, the analytical determination of the test substance concentrations was performed in each test concentrations at the beginning and at the end of the test.

For all evaluations and results were used the concentrations measured at the beginning of the test.

Test results:

24h – LC₅₀ > 128 mg·L^-1 (determined concentration)

48h – LC₅₀ > 128 mg·L^-1 (determined concentration)

72h – LC₅₀ > 128 mg·L^-1 (determined concentration)

96h – LC₅₀ > 128 mg·L^-1 (determined concentration)

24h – NOEC = 128 mg·L^-1 (determined concentration)

48h – NOEC = 128 mg·L^-1 (determined concentration)

72h – NOEC = 128 mg·L^-1 (determined concentration)

96h – NOEC = 128 mg·L^-1 (determined concentration)

24h – LC₁₀₀ > 128 mg·L^-1 (determined concentration)

48h – LC₁₀₀ > 128 mg·L^-1 (determined concentration)

72h – LC₁₀₀ > 128 mg·L^-1 (determined concentration)

96h – LC₁₀₀ > 128 mg·L^-1 (determined concentration)