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Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 September - 24 September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

The applied dose could not be analytically verified successfully.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Test material: Harpin fermentation extract (Harpin-αβ extract)
- Batch No. of test material: R134-1
- Appearance: Liquid/ tan, light brown
- Active ingredient content: 0.401% w/w
- Storage conditions: On dry ice (≤ -70 °C), dark, dry

Analytical monitoring:

yes

Details on sampling:

Analytical samples (100 mL in 250 mL Schott bottles) were taken from the test item concentration and control at test start and after 24 hours from fresh and aged solutions and after 48 hours from aged solutions. For each sampling also a retain sample was taken. Additionally a sample of the test item in pure form was taken.
All samples were stored refrigerated until they were transferred to the analytical laboratory.

Vehicle:

no

Details on test solutions:

The necessary amount of test item for preparing the stock solution S1 was weighed on a weighing scoop and transferred to a volumetric flask. Test medium was added up to the bench mark and the solution was homogenised by shaking. Afterwards the solution was clear and transparent. The preparation procedure was repeated after 24 hours. At least 50 mL of the prepared solutions were transferred to each test vessel

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna Straus
- Strain/clone: Clone V
- Source: The animals are continuously bred in the laboratory and were originally purchased in a healthy condition from the Federal Environment Agency in Berlin/Germany.
- Age: Freshly hatched daphnids less than 24 hours old were used for the test.
- Feeding during test: No

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

Total hardness: 13 dH (232 mg/L as CaCO3)

Test temperature:

19.4 - 21.0°C

pH:

Initial pH of control: 7.87
Test: 7.77 - 7.96

Dissolved oxygen:

Initial: 8.9 mg/L
Test: ≥ 8.7 mg/L

Nominal and measured concentrations:

Nominal concentration: 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass vessel
- Type: closed (covered with a glass plate)
- Material, size, headspace, fill volume: 100 mL glass vessels were filled up with > 50 mL test solution and covered with a glass plate (thus reducing evaporation)
- Aeration: None
- No. of organisms per vessel: 5 daphnia/ vessel
- No. of vessels per concentration (replicates): 4 replicates per treatment
- No. of vessels per control (replicates): 4 replicates for control
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: >10 mL of test solution for each animal

TEST MEDIUM / WATER PARAMETERS
- Medium: Elendt M4 medium

OTHER TEST CONDITIONS
- Adjustment of pH: Not specified
- Photoperiod: 16 hours photoperiod /8 hours darkness daily
- Light intensity: Not specified

EFFECT PARAMETERS MEASURED: After 24 h and 48 h the immobilised daphnids were counted. All daphnids not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobilised. If present, behavioural changes of daphnids were recorded at 24 and 48 hours after starting the test

VEHICLE CONTROL PERFORMED: No

Reference substance (positive control):

yes

Remarks:

Two concentrations of the reference item potassium dichromate (1.00 mg/L, 2.00 mg/L) were tested around the same time period as the study

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Biological results:
After 24 and 48 hours of exposure no immobilisation was observed in the control and the test item concentration 100 mg/L.
No behavioural changes could be observed.

Analytical results:
The initial measured content of TOC was below LOD (LOD = 0.5 mg/L; measured value 307 µg/L (0 hours fresh) and 390 µg/L (24 hours fresh)) at 100 mg/L. In the aged sample the measured TOC was below LOD (measured value 363 µg/L) at 100 mg/L. The measured TOC concentrations might result from the test medium, since the TOC content of the solution of 100 mg/L in deionized water was determined to be below LOD. Furthermore, the measured concentrations of TOC were of similar values in the test item concentration as corresponding results from the control media measured values, in both the fresh and aged analysed samples.
Due to the properties and the composition of the test item no analytical dose verification was possible with commonly applied analytical methods. Therefore the toxicological endpoints were evaluated using nominal concentrations. Due to correct routinely handling of the test item and the test item solutions, a correct application of the test item is assumed.

Results with reference substance (positive control):

The results indicate an EC50 (24 h) of the reference item potassium dichromate between 1.00 and 2.00 mg/L. Since the results are in accordance with the requirements of the OECD guideline 202 and fall within the historical data generated with the reference item at the testing facility, the daphnids were suitable for the determination of the toxicological effects of the test item.

Table1:         Results of the test, 24 h values

	Nominal test item concentration [mg/L]
	Control	100
	Immobilised daphnids after 24 h
Replicate 1	0	0
Replicate 2	0	0
Replicate 3	0	0
Replicate 4	0	0
Σ	0	0
%	0	0

Table 2:         Results of the test, 48 h values

	Nominal test item concentration [mg/L]
	Control	100
	Immobilised daphnids after 48 h
Replicate 1	0	0
Replicate 2	0	0
Replicate 3	0	0
Replicate 4	0	0
Σ	0	0
%	0	0

Table 3:         Determined concentration ofTOC in the test samples

Test item nominal [mg/L]		Harpin-αβ nominal [mg/L]	Sampling	TOC [µg/L]	% Standard Deviation
Control	0		0 fresh	< LOD (265)*	6.94
			24 fresh	< LOD (263)*	3.02
			48 aged	< LOD (496)*	1.38
100	0.4		0 fresh	< LOD (307)*	0.13
			24 fresh	< LOD (390)*	0.50
			48 aged	< LOD (363)*	6.00

Limit of detection (LOD) = 0.5 mg/L TOC

*value results from the test medium (containing vitamins and other organic carbons)

 

Table 4:         Determined of the TOC content of the test item as a bulk material

Test item nominal [mg/L]		Harpin-αβ nominal [mg/L]	TOC [µg/L^1)]
100	0.4		0*

* below Limit of detection (LOD = 0.5 mg/L TOC)

¹⁾Determined in deionized water

Validity criteria fulfilled:

yes

Remarks:

In the study the control immobilisation was 0% (should be ≤ 10 %). The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in all test units. In this test, the dissolved oxygen concentration at the end of the test was ≥ 8.7 mg/L.

Conclusions:

According to the results of the test, the EC50 (48 h) for immobilisation was estimated to be > 100 mg/L (nominal). The corresponding NOEC (48 h) was 100 mg/L (nominal).

Description of key information

A GLP study was performed in accordance with OECD Guideline 202 to assess the toxicity of the test material, Harpin fermentation extract, to Daphnia magna. According to the results of the test, the EC50 (48 h) was supposed to be > 100 mg/L (nominal). The corresponding NOEC (48 h) was 100 mg/L (nominal).

Additional information

The study performed on EBC 351 (1% Harpin ab), supports the lack of toxicity of 'Cell Free Harpin Extract of Harpinαβ produced by fermentation' and that classification is not required.