Humic acids, sodium salts

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04.11.2019 - 18.12.2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

Adopted: October 2, 2012

Deviations:

yes

Remarks:

see Any other information

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

Molecular formula:

NA

IUPAC Name:

Humic acid potassium salts

Test material form:

solid: flakes

Remarks:

black-brown

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.: 25062019/3
- Expiration date of the lot/batch: 06/2024
- Stability under test conditions: stable
- Storage condition of test material: dry conditions

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Preliminary test: The analytical determination of the test item concentrations was performed once a week in the preparation of solutions (fresh solutions) and in the renewal of solutions (old solutions). The samples for analysis were taken from the highest (10 mg·L-1) and the lowest test concentration (1.0 mg·L-1). The samples for analysis (0 hours – fresh solution) were prepared once a week and immediately delivered in transport box to analytical laboratory. The samples for analysis in renewal (old solution) were taken in defined time intervals and delivered in transport box to analytical laboratory.

Definitive test: The analytical determination of the test item concentrations was performed once a week in the preparation of solutions (fresh solutions) and in the renewal of solutions (old solutions). The samples for analysis were taken from the concentrations 10 mg·L-1, 5 mg·L-1 and 1.2 mg·L-1. The samples for analysis (0 hours – fresh solution) were prepared once a week and immediately delivered in transport box to analytical laboratory. The samples for analysis in renewal (old solution) were taken in defined time intervals and delivered in transport box to analytical laboratory.

Sample storage conditions before analysis: all samples were stored at laboratory temperature. The samples were analyzed in the day of delivery.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The stock solution of the test item was prepared in M4 medium. 20 mg of the test item was weighed into 2000 mL of the M4 medium for the preliminary test and 50 mg of the test item was weighed into 1000 mL of the M4 medium for the definitive test. The fresh stock solutions were prepared three times per week.

The concentrations of solutions used in the preliminary and the definitive test were obtained by dilution of the stock solution with M4 medium.
Preliminary test: 10, 5, 1, 0 mg·L-1
Definitive test: 10, 5.9, 3.5, 2.1, 1.2, 0 mg·L-1

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM

Daphnia magna Straus
- Source, method: aperiodic parthenogenesis from own laboratory breeding
The grow-up gravid females are separated into M4 medium. Young daphnia born in 24 hours are segregated by observation under a microscope. Only the females are transferred to the test containers.
- Feeding during test, type, amount, frequency:
daily (out of 09.11., 10.11., 16.11., 17.11., 23.11., 24.11. in preliminary test and 30.11., 1.12., 07.12., 08.12., 14.12., 15.12. in the definitive test), the mixture of two algae (Desmodesmus subspicatus and Raphidocelis subcapitata 1:1), ration levels 0.5 mg C/daphnia/day at all times.
Volume of the mixture of algae for feeding: 430 µL/100 mL in preliminary test and 570 µL/100 mL in definitive test

Study design

Test type:

semi-static

Water media type:

freshwater

Remarks:

The stock solutions of the M4 medium were prepared by adding measured amounts of the individual chemicals to deionized water.

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

none

Test conditions

Hardness:

Preliminary test: 2.44 – 2.51 mmol Ca+Mg /L
Definitive test: 2.43 – 2.49 mmol Ca+Mg /L

Test temperature:

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

pH:

Preliminary test: 8.1 – 8.5
Definitive test: 8.0 – 8.5

Dissolved oxygen:

Preliminary test: 7.5 – 8.5
Definitive test: 7.1 – 8.6

Salinity:

not applicable

Conductivity:

Preliminary test: 1.22 – 2.60 µS·cm-1
Definitive test: 1.54 – 1.98 µS·cm-1

Nominal and measured concentrations:

Preliminary test: 10, 5, 1 mg·L-1
Definitive test: 10, 5.9, 3.5, 2.1, 1.2, 0 mg·L-1

Details on test conditions:

TEST SYSTEM
- Material, size, headspace, fill volume: 100 ml
- Aeration: without
- Renewal rate of test solution (frequency/flow rate): 3× per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Volume of the mixture of algae for feeding: 430 µL/100 mL in preliminary test and 570 µL/100 mL in definitive test

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water
- Test medium: M4
- Conductivity of the deionized water:
Preliminary test: 1.22 – 2.60 µS·cm-1
Definitive test: 1.54 – 1.98 µS·cm-1

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light
- Light intensity: 1568 – 1575 lx (not exceeding 1250 – 1670 lx in general)

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 2

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

The following data were recorded during the test: numbers of living offspring by each parent animal, presence of dead offspring and mortality parent animals. The total number of offspring per parent animal was calculated for each test vessel. If in any test vessel the parent animal died during the test, then the test vessel was excluded from the analysis.
The mean reproductive output for each concentration and control were calculated. Reduction of reproductive output was expressed in %.

- Mortality of control: 0 parent animal; 0 offspring

Results with reference substance (positive control):

a) Reference test
The sensitivity of the test species and correctness of test performance is periodically verified by testing with the reference substance, potassium dichromate. The results of the verification test with K2Cr2O7 are the following:
21 days – EC50 = 0.26 mg/L (95% confidence limit: 0.25 – 0.27 mg/L)

b) Data in the literature
HERMENS, JOOP, et al. Joint effects of a mixture of 14 chemicals on mortality and inhibition of reproduction of Daphnia magna. Aquatic Toxicology, Volume 5, Issue 4, November 1984, Pages 315-322.
21 days – EC50 = 0.27 mg/L

c) Method validity evaluation
The reduction in reproductive output of the test organism by the reference substance (21d – EC50) obtained from our reference test is conformable to the values stated in the literature (Hermens et al., 1984).

Reported statistics and error estimates:

Analytical method (spectrophotometric method) - Data acquisitions and data processing were performed by using of the WinAspect Plus Version: 4.2.0.0. Statistical assessment of experimental data was carried out in Excel (Microsoft Corporation).
The value of EC50 and confidence intervals of the EC50 were calculated using the software ToxRat Professional Version 3.2.1.

Any other information on results incl. tables

The total number of living and dead daphnia

Concentration mg·L^-1	Living offspring Total	Living offspring of survived parents	Dead parent animals	Dead offspring Total
10	156	156	0	0
5.9	224	224	0	0
3.5	305	305	0	0
2.1	350	350	0	0
1.2	411	411	0	0
K	602	602	0	0

The evaluation of reduction in reproductive output of daphnia

Concentration mg·L^-1	The mean number of the living offspring/daphnia	Reduction in reproductive output versus control %
10	15.6	74.1 %
5.9	22.4	62.8 %
3.5	30.5	49.3 %
2.1	35.0	41.9 %
1.2	41.1	31.7 %
K	60.2	-

Detailed information on offspring per individual replicate, concentration-response curve and results of chemical analyses are provided by file in section Attached bacround material.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see Overall remarks

Conclusions:

The nominal concentrations of the test item were used for all evaluations and results.
21 days – EC50 = 3.2 mg/L (95% confidence limit: 1.9 – 5.2 mg·L-1)
21 days – EC10 = 0.3 mg/L (95% confidence limit: 0.2 - 0.4 mg·L-1)
Approach function: Non-linear Regression
21 days – NOEC < 1.2 mg·L-1, resp. 21 days – NOEC = 1.2 mg·L-1 for CSA

Executive summary:

The test item, Humic acids, potassium salts, was tested in reproduction test on Daphnia magna.

The test was performed according to OECD Test Guideline No. 211 - Daphnia magna Reproduction Test. Adopted: October 2, 2012.

 

The preliminary test was performed in a range of the test item concentrations 1 – 10 mg·L-1. The test was performed as semi-static, frequency of the test solutions renewal was three times a week.

The highest calculated reduction in reproductive output was 74.9 %. On the basis of the preliminary test results the definitive test was performed.

The definitive test was performed in a range of the test item concentrations 1.2 – 10 mg·L-1. The test was performed as semi-static, frequency of the test solutions renewal was three times a week.

 

The samples for analytical determination were taken once a week in the preparation of solutions (fresh solutions) and in the renewal of solutions (old solutions).

 

For all evaluations and results the nominal concentrations of the test item were used.

Test results, based on nominal concentration:

21 days – EC50 = 3.2 mg/L (95% confidence limit: 1.9 – 5.2 mg·L-1)

21 days – EC10 = 0.3 mg/L (95% confidence limit: 0.2 - 0.4 mg·L-1)

Approach function: Non-linear Regression

21 days – NOEC < 1.2 mg·L-1