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 aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

key study

Study period:

from October 1, 2007 to November 20, 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.2 (Acute Toxicity for Daphnia)

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. 100 mg of the test substance was weighed into 1000 mL of the dilution water for preliminary test and 200 mg of the test substance was weighed into 1000 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):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: daphnia
- Strain: Daphnia magna Straus
- Source: own laboratory breeding
- Age at study initiation (mean and range, SD): < 24 h
- Method of breeding:
The grow-up gravid females are separated into dilution water. Young dapdhnia born in 24 hours are segregated to the handling container from
which they entered the experiment. At handling a care should be taken of not to penetrate the air under animalś shell.
- Feeding during test: no
- Aeration during test: no

Study design

Test type:

static

Water media type:

freshwater

Total exposure duration:

48 h

Post exposure observation period:

none

Test conditions

Hardness:

Content of (Ca+Mg) in dilution water: 2.63 mmol.L-1 (preliminary test)
Content of (Ca+Mg) in dilution water: 2.60 mmol.L-1 (limit test)

Test temperature:

20±2 °C, with a variation of ±1°C at each individual test

pH:

pH of dilution water: 7.9
pH during the test: 7.8 - 8.0

Dissolved oxygen:

Oxygen content at the beginning of test: 8.7 - 8.9 mg.L-1 at temperature of 19.7ºC
Oxygen content at the end of test: 8.5 - 8.7 mg.L-1 at temperature of 19.6ºC

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open baker
- Material, size, headspace, fill volume: glas
- Aeration: no
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2


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 into 1000 mL of distilled water.
A. 117.6 g CaCl2.2H20 in 1 L of distilled water
B. 49.3 g MgSO4.7H2O in 1 L of distilled water
C. 25.9 g NaHCO3 in 1 L of distilled water
D. 2.3g KCl in 1 L of distilled water
The dilution water should be of pH 7.8 ± 0.2.
The sum of Ca + Mg ions should be approx. 2.5 mmol.L-1.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Lighting: natural variation of daily light and dark
- Light intensity: daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilization of daphnia

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: natural daily light and dark
- Light intensity:

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 116 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 116 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

116 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

116 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 5

Duration:

24 h

Dose descriptor:

LC100

Effect conc.:

> 116 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 6

Duration:

48 h

Dose descriptor:

LC100

Effect conc.:

> 116 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

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 concentration. 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 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 determined at the beginning of the test.

Results with reference substance (positive control):

- Results with reference substance valid? yes
The results of the verification test with K2Cr2O7, carried out in period from 12.9. to 14.9. 2007 were following:

24h – EC50 = 2.28 mg.L-1 (95 % confidence limit: 2.13 – 2.43 mg.L-1)
48h – EC50 = 1.06 mg.L-1 (95 % confidence limit: 0.76 – 1.36 mg.L-1)
The result 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 was:
48-hour EC50 = 0.61 – 1.11 mg.L-1
The immobilisation of test species in reference substance solution expressed as EC50 fits into the accepted range in the interlaboratory tests.

Reported statistics and error estimates:

No immobilisation was observed in limit test. Therefore, exact values of EC50 could not be calculated and the values of EC are given in the form of a range.
The NOEC values are identified by direct observation.
For all evaluations and results were used the concentrations determined at the beginning of the test.

Any other information on results incl. tables

Immobilisation of daphnia – limit test

Determined concentration	mg·L^-1	116	98.7	79.4	66.9	57.0	47.2	C
Nominal concentration	mg·L^-1	150	125	104	87	73	61	C
Number of live animals	24 hours/1^st series	10	10	10	10	10	10	10
Number of live animals	24 hours/2^nd series	10	10	10	10	10	10	10
Number of live animals	24 hours/total	20	20	20	20	20	20	20
Immobilisation %	24 hours	0	0	0	0	0	0	0
Number of live animals	48 hours/1^st series	10	10	10	10	10	10	10
Number of live animals	48 hours/2^nd series	10	10	10	10	10	10	10
Number of live animals	48 hours/total	20	20	20	20	20	20	20
Immobilisation %	48 hours	0	0	0	0	0	0	0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Executive summary:

The test substance was tested in acute immobilisation test on daphnia.

The test was performed according to EU method C.2. - Acute toxicity for Daphnia, Directive 92/69/EEC, published in O.J. L 383A, 1992.

The water solubility of the test substance is 279 g·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 concentration. 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 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 determined at the beginning of the test.

Test results:

24–hour EC₅₀ > 116 mg·L^-1 (determined concentration)

48–hour EC₅₀ > 116mg·L^-1 (determined concentration)

24–hour NOEC = 116 mg·L^-1 (determined concentration)

48–hour NOEC = 116 mg·L^-1 (determined concentration)

24–hour EC₁₀₀ > 116 mg·L^-1 (determined concentration)

48–hour EC₁₀₀ > 116 mg·L^-1 (determined concentration)