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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

key study

Study period:

from October 12, 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.3 (Algal Inhibition test)

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:

The analytical determination of the test substance concentrations was performed at the beginning and at the end of the test. The samples for analysis were taken from the highest and the lowest test concentration and from the highest test concentration without algae. The samples for analysis (0 hours) were prepared at the beginning of the test and immediately delivered in transport box to analytical laboratory. The samples were analysed on the same day. The samples for analysis at the end of the test (72 hours) were delivered to analytical laboratory immediately after the end of testing. The samples were analysed on the same day. All samples were stored at laboratory temperature.

Test solutions

Vehicle:

no

Details on test solutions:

The test substance was soluble in the test medium in concentration needed for the test. The stock solution of the test substance was prepared in test medium. 100 mg of the test substance was weighed into 1000 mL of test medium for preliminary and full test. The concentrations of solutions used in the preliminary and full test were obtained by dilution of the stock solution with test medium. The exact concentrations were measured.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The strain culture was preinoculated from the stock solution and cultivated in flasks with the test medium on indirect daylight at laboratory temperature. Algae inoculum for the test was sampled from exponentially growing inoculum culture. The strain culture was always set to pre-culturing of cells for 3 days before the start of the test. Inoculum culture was kept 3 days under conditions at which the test was performed. After this period the culture was in the state of the exponential growth and had the cell density suitable for the performance of the test. The cell density of the pre-culture was measured just before the start of the test and the needed inoculum volume was calculated.

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: Desmodesmus subspicatus Brinkmann 1953/SAG 86.81
- Source (laboratory, culture collection): obtained from the collection of autotrophic organisms of The Botanic Institute of the Czech Academy of Science, Třeboň, on date 13.9.2007

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Test conditions

Test temperature:

Temperature during the test: 23 - 23.5 °C

pH:

pH of test medium: 7.7

Nominal and measured concentrations:

Test concentrations: 100, 77, 59, 45, 35, 27 and 21 mg in 1000 mL of test medium

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyers’ flasks with stoppers venting air
- Material, size, headspace, fill volume: glass, fill volume 50 mL
- Aeration: without
- Initial cells density: 10000 cells in 1 mL of the test solution
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
Preparation of test medium
The test medium was prepared by admixing 10 mL of basic stock solution A and 1 mL of basic stock solutions B, C, D in 1000 mL distilled water.
Solution A Substance Content Unit
NH4Cl 1.5 g
MgCl2.6H20 1.2 g
CaCl2.2H2O 1.8 g
MgSO4.7H2O 1.5 g
KH2PO4 0.16 g
Water up to 1000 mL
Solution B Substance Content Unit
FeCl3.6H2O 80 mg
Na2EDTA.2H2O 100 mg
Water up to 1000 mL
Solution C Substance Content Unit
H3BO3 185 mg
MnCl2.4H2O 415 mg
ZnCl2 3.0 mg
CoCl2.6H2O 1.5 mg
CuCl2.2H2O 0.01 mg
Na2MoO4.2H2O 7.0 mg
Water up to 1000 mL
Solution D Substance Content Unit
NaHCO3 50 g
Water up to 1000 mL

Conductivity of distilled water: 0.116 mS.m-1
All chemicals used were of analytical-grade.

OTHER TEST CONDITIONS
- Lighting: continuous, min 6000 lux, max. 10000 lux,
- Volume of tested mixture: 50 mL
- Other: Agitation of algae culture by shaking

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: counting chamber

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

87 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 78.11-95.92 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

77 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

77 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Test quality criteria
The quality criteria are given in the guideline.
The test should satisfy the following quality criteria:
1) The density of cells should increase minimally 16 times during 72 hours in the control culture.
2) The pH of the controls should not normally deviate more than 1.5 unit during the test.
3) The test substance concentration in test solutions should not drop under 80 % of input one during of the test.

The test quality criteria at point 1, 2 and 3 were fulfilled.

Results with reference substance (positive control):

The sensitivity of the test species and correctness of test performance was periodically verified on a three-month period by testing the reference substance, potassium dichromate.
The results of the verification test with K2Cr2O7, carried out in period from 10.9. to 13.9.2007, were following:
72h – EbC50 = 0.35 mg.L-1 (95 % confidence limit: 0.30 – 0.39 mg.L-1)
72h – ErC50 = 0.67 mg.L-1 (95 % confidence limit: 0.60 – 0.75 mg.L-1)

The given results are convenient to range of the results of the interlaboratory tests. The results of the last interlaboratory test arranged by CSlab spol. s.r.o. in 2007 for potassium dichromate were:
72 hour - EbC50 = 0.28 – 0.38 mg.L-1
72 hour - ErC50 = 0.67 – 1.06 mg.L-1
The inhibition of test organisms in the reference substance solution meets criteria determined by the interlaboratory tests.

Reported statistics and error estimates:

Test evaluation
The value of EbC50 was calculated by means of software “Toxicita”(product of Water Research Institute, Ostrava, Czech Republic). The determination of NOEC values was done by ANOVA (Analysis of Variance) analysis. Used ANOVA method is the part of statistical software QC.Expert 2.5 © 1998-2000 (TriloByte, Czech Republic).

Any other information on results incl. tables

Mean cell density full test

Nominal concentration mg·L^-1	Mean cell density in 1 mL
	0 hours	24 hours	48 hours	72 hours
100	10000	39583	95833	429167
77	10000	47917	120833	897917
59	10000	54167	143750	1072917
45	10000	66667	152083	1154167
35	10000	70833	152083	1347917
27	10000	77083	214583	1275000
21	10000	87500	204167	1375000
Control	10000	100000	208333	1331250

 Percentual reduction of growth rate and percentual inhibition of cell growth – full test

Nominal concentration mg·L^-1	Growth rate		Cell growth
	Mean growth	Reduction %	Mean area A	Inhibition %
100	1.25	23.1	325000	65.8
77	1.50	8.1	592708	37.5
59	1.56	4.4	709375	25.2
45	1.58	2.9	770833	18.8
35	1.63	-0.3	871875	8.1
27	1.62	0.9	904167	4.7
21	1.64	-0.7	954167	-0.5
Control	1.63	-	948958	-

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The results of the test were equivocal.
The value 72h - EbC50 = 87 mg/L (50% reduction of growth) suggests to specify the substance as harmful to aquatic organisms.
The 72h - ErC50 > 100 mg/L (50% reduction of ) does not suggests to specify the substance as harmful to aquatic organisms.
This fact can be caused by colour of the test solution (see point 5.2.1.3 of Directive 67/548/EEC - Annex VI). In this case - when algae growth is inhibited solely as a result of a reduction in light intensity - the 72h-IC50 for algae should not be used as a basis for classification.

Analytical method for the determination of substance stability in dilution water: spectrofotometry at 300 nm

Executive summary:

The test substance was tested for growth inhibition on algae.

The test was performed according to EU Method C.3, Algal inhibition test, Directive 92/69/EEC, Published in OJ 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 test substance Humic acids, potassium salts was stable in test medium in conditions of the test. Based on toxicity of the test substance found in the preliminary test, the full test was performed in appropriate concentration range.

The full test was performed in range of test substance nominal concentrations 21 - 100 mg·L^-1.

The nominal concentrations of the test substance were used for all evaluations and results.

Test results:

72 hour - E_bC₅₀ =  87 mg·L^-1 (95% confidence limit: 78.11 – 95.92 mg·L^-1) (nominal concentration)

72 hour - E_rC₅₀ > 100 mg·L^-1(nominal concentration)

72 hour - NOEC_b = 77 mg·L^-1 (nominal concentration)

72 hour - NOEC_r = 77 mg·L^-1 (nominal concentration)