Registration Dossier

Administrative data

Endpoint:
long-term toxicity to fish, other
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
The chemical safety assessment according to Annex I indicates the need to investigate further the effects on aquatic organisms. Long-term toxicity test on invertebrates (Daphnia) was performed for source substance Humic acids potassium salts. The result of this test shows no toxicity based on long-term exposure. The results of short-term toxicity tests (fish, invertebrates, algae) for source substance show also no toxicity. Based on results of aquatic toxicity tests for Humic acids sodium salts no classification is required as well.
Based on all results the performing of long-term toxicity study on fish is scientifically not necessary. Moreover, this would mean unnecessary testing on vertebrates.
Cross-reference
Reason / purpose for cross-reference:
data waiving: supporting information
Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
04.11.2019 - 18.12.2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable guideline study, tested with the source substance Humic acids, potassium salts. According to the ECHA guidance “Practical guide 6 the reliability was changed to RL2 to reflect the f act that this study was conducted on a read-across substance.
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
See attached document in IUCLID Section 13 - Read across_fin

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See attached document in IUCLID Section 13 - Read across_fin

3. ANALOGUE APPROACH JUSTIFICATION
Using a technique Read across to predict endpoint information for Humic acids, sodium salts by using data from the same endpoint from source chemicals – Humic acids, potassium salts is appropriate based on similarities in general molecular structure of humic acids (the source of substances) as such and functionality of their water soluble salts. Production of sodium or potassium salts is only necessary for increase of bioavailability of humic acids. Given that both the source substance and the target substance is produced entirely in the same manner (source of oxyhumolite for both registered substances is the same and substances are manufactured exactly in the same manner) and
have comparable results in available studies, which did not lead to classification, it is expected that the results from long-term study on the target substance will not provide significantly different information. Therefore it is concluded as unnecessary to perform long-term toxicity study for Humic acids, sodium salts.

4. DATA MATRIX
See attached document in IUCLID Section 13 - Table 1: Data matrix
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
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)
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.


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 (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
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
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
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
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.

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.

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

Data source

Materials and methods

Results and discussion

Applicant's summary and conclusion