Escherichia coli dehalogenase catalyst

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10th September 2018 to 29th October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Unknown 6

IUPAC Name:

Unknown 6

Constituent 2

Reference substance name:

Unknown 7

IUPAC Name:

Unknown 7

Constituent 3

Reference substance name:

Unknown 5

IUPAC Name:

Unknown 5

Constituent 4

Reference substance name:

Unknown 4

IUPAC Name:

Unknown 4

Constituent 5

Reference substance name:

Unknown 3

IUPAC Name:

Unknown 3

Constituent 6

Reference substance name:

Unknown 2

IUPAC Name:

Unknown 2

Constituent 7

Reference substance name:

Unknown 1

IUPAC Name:

Unknown 1

Test material form:

solid

Specific details on test material used for the study:

- Source and lot/batch No.of test material: 4898318
- Expiration date of the lot/batch: 01 Mar 2019
- Purity test date: Not specified
- Storage condition of test material: deep frozen (≤ -18 °C), dark, dry

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All samples were measured without dilution.
- Sampling method: Analytical samples (100 in 250 mL Schott bottles) were taken from the test item concentration and control at test and after 48 hours from aged solutions.
- Sample storage conditions before analysis: All samples were stored refrigerated until they were transferred to the analytical laboratory.

Test solutions

Vehicle:

no

Details on test solutions:

The following nominal concentrations were tested in the main test: 100, 50.0, 25.0, 12.5, 6.25 mg/L, and control.
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 (Elendt M4 medium) was added up to the bench mark and the solution was homogenised by shaking. Afterwards the solution was clear and transparent and foaming was observed. Lower test solutions were prepared by dilution of the appropriate solution with test medium. > 50 mL of the prepared solutions were transferred to each test vessel. See table below under 'Any other information on materials and methods incl. tables'.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna Straus
- Strain/clone: Clone V
- Justification for species other than prescribed by test guideline: Not specified.
- Source: The animals were continuously bred in the laboratory and were originally purchased in a healthy condition from the Federal Environment Agency in Berlin/Germany.
- Age of parental stock (mean and range, SD): Not specified
- Feeding during test: Yes
- Food type: single cell green algae (Desmodesmus subspicatus, formerly Scenedesmus subspicatus)
- Amount: Not specified
- Frequency: At least three times a week

Daphnia magna was bred as single culture (1 daphnid per 100 mL) in Elendt M4 medium. The pH-value of the aerated water was within a range of 6.0 – 9.0. The dissolved oxygen was above 60 % saturation and the total hardness 140 - 250 mg/L (as CaCO3), corresponding to 7.8 - 14°dH.
The daphnids were reared at a temperature of 20 ± 2 °C in a climatic chamber with 16 hours of illumination and 8 hours of darkness. The medium was changed three times per week. A pipette was used to separate the young daphnids from the adults.
Freshly hatched daphnids less than 24 hours old were used for the test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

The total hardness was 13°dH (232 mg/L as CaCO3).

Test temperature:

19.7 – 20.7 °C

pH:

At test start, the pH-value of the control (untreated test medium) was 7.82

Dissolved oxygen:

At test start, the dissolved oxygen concentration was 8.9 mg/L

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal concentrations of 100, 50.0, 25.0, 12.5, 6.25 mg/L.
The initial measured TOC content was between 71 and 95 % of nominal and the aged measured content was between 35 and 101 % of nominal.

Details on test conditions:

TEST SYSTEM
- Test vessel: four 100 mL glass beakers per treatment, each filled with > 50 mL, one additional replicate for physico - chemical measurements without test organisms.
- Material, size, headspace, fill volume: Glass vessel (100 mL), were filled up with > 50 mL test solution. The test units were covered with a glass plate (thus reducing evaporation).
- Aeration: None
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- No. of organisms: 20 per treatment in 4 replicates of 5

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

EFFECT PARAMETERS MEASURED: After 24 h and 48 h the immobilised daphnids were counted.

VEHICLE CONTROL PERFORMED: Untreated test medium

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

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

Details on results:

- Behavioural abnormalities: No behavioural changes could be observed.
- Observations on body length and weight: Not evaluated
- Other biological observations: None
- Mortality of control: None
- Other adverse effects control: None
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: After 24 hours at test item concentrations 12.5 to 100 mg/L an increasing amount of substance could be observed at the bottom of the test vessels. Additionally from 25.0 to 100 mg/L turbidity of the aqueous phase increased. After 48 hours similar observations were made as after 24 hours. However, substance at the bottom of the test vessels was already observed at 6.25 mg/L.

The initial measured TOC content was between 71 and 95 % of nominal and the aged measured content was between 35 and 101 % of nominal. Since the TOC content in the initial samples was between 80 and 120 % of nominal the toxicological endpoints were evaluated using nominal concentrations.

Results with reference substance (positive control):

See table below for details.
In order to check the validity of the results, the toxicity of the reference item potassium dichromate was tested at 1.00 and 2.00 mg/L with 20 test organisms per test concentration.

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.

Reported statistics and error estimates:

EC50 and NOEC were evaluated using nominal concentrations.
Due to an immobilisation below 50% the database was weak for probit analysis which hence was not performed.

Any other information on results incl. tables

Results of the toxic reference test, started on 11 Sep 2018:

K2Cr2O7	24 h			48 h
[mg/L]	1.00	2.00		1.00		2.00
	immobilised daphnids
Replicate 1	1		5	5	5
Replicate 2	2		5	4	5
Replicate 3	0		5	3	5
Replicate 4	4		5	5	5
S	7		20	17	20
%	35		100	85	100

Biological results:

Table 1: Results of the test, 24 h values:

	Nominal test item concentration [mg/L]
	Control	6.25	12.5	25.0	50.0	100
	Immobilised daphnids after 24 h
Replicate 1	0	0	0	0	0	0
Replicate 2	0	0	0	0	0	0
Replicate 3	0	0	0	0	0	0
Replicate 4	0	0	0	0	0	0
Σ	0	0	0	0	0	0
%	0	0	0	0	0	0

Table 2: Results of the test, 48 h values:

	Nominal test item concentration [mg/L]
	Control	6.25	12.5	25.0	50.0	100
	Immobilised daphnids after 48 h
Replicate 1	0	0	1	0	0	1
Replicate 2	0	0	0	0	0	1
Replicate 3	0	0	0	0	0	1
Replicate 4	0	0	0	0	0	0
Σ	0	0	1	0	0	3
%	0	0	5	0	0	15

Table 3: Temperature of the test solutions

	nominal test item concentration [mg/L]
	Control	6.25	12.5	25.0	50.0	100
Time [h]	Temperature [°C]
0 fresh	20.4	20.7	20.7	20.7	20.7	20.7
24 aged	19.9	19.8	19.7	19.9	19.9	20.2
48 aged	20.3	20.1	20.0	20.1	20.2	20.3
Mean	20.2	20.2	20.1	20.2	20.3	20.4
Std. dev.	0.3	0.5	0.5	0.4	0.4	0.3
Mean	20.2
Std. dev.	0.3

Table 4: pH-values of the tests solutions

	nominal test item concentration [mg/L]
	Control	6.25	12.5	25.0	50.0	100
Time [h]	pH
0 fresh	7.82	7.94	7.94	7.94	7.93	7.91
24 aged	7.85	7.88	7.88	7.83	7.77	7.57
48 aged	7.91	7.86	7.75	7.63	7.56	7.60
Mean	7.86	7.89	7.86	7.80	7.75	7.69
Std. dev.	0.05	0.04	0.10	0.16	0.19	0.19
Mean	7.81
Std. dev.	0.13

Table 5: O2 concentration of the test solutions

	nominal test item concentration [mg/L]
	Control	6.25	12.5	25.0	50.0	100
Time [h]	Oxygen [mg/L]
0 fresh	8.9	8.9	8.9	8.9	8.9	8.9
24 aged	9.0	8.7	8.7	8.4	8.0	6.5
48 aged	8.5	8.2	7.8	7.3	7.0	6.8
Mean	8.8	8.6	8.5	8.2	8.0	7.4
Std. dev.	0.3	0.4	0.6	0.8	1.0	1.3
Mean	8.2
Std. dev.	0.8

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

According to the results of the test, the EC50 (48 h) for immobilisation is > 100 mg/L (nominal). The corresponding NOEC (48 h) was 50.0 mg/L (nominal).

Executive summary:

Study Objective:

The objectives of this study were to determine the immobilisation effect of the test item Escherichia coli dehalogenase catalyst on the water flea Daphnia magna under worst-case exposure conditions, the no observed effect concentration (NOEC) and the effect median concentration (EC₅₀), was determined where possible.

Materials and methods:

Test item: Escherichia coli dehalogenase catalyst, batch number: 4898318, active ingredient (a.i.): (R)-2-haloacid halidohydrolase, activity of dehalogenase.: 120 U/g.

Test organisms: Daphnia magna Straus, Clone V, max. 24 hours old.

Test design: Static dose-response test with twenty test organisms per treatment (4 replicates of 5 test organisms each) were used. The duration of the test was 48 hours.

Endpoints: Endpoints reported are the EC50 and the NOEC after 24 and 48 hours.

Test rates: A static main test with nominal concentrations of 100, 50.0, 25.0, 12.5, 6.25 mg/L, and control was performed.

Test conditions: Temperature, pH-value and oxygen concentration of the test solutions measured after 0, 24 hours and 48 hours are reported. Hardness of the test medium (untreated control) was measured on the day of application.

Samples analysed: Analytical samples taken at 0 hours (initial value) and 48 hours from aged test solutions were analysed from control and all test item concentrations.

Statistics: The values for EC50 were not determined statistically. The NOEC was established based on the highest concentration at which the immobilisation is not higher than the allowed control immobilisation (≤ 10 % immobilisation).

Validity criteria: Control immobilisation: The percentage of immobilisation should be ≤ 10 %. In this study the control immobilisation was 0 %.

Oxygen concentration: 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 ≥ 6.5 mg/L.

Test conditions: The total hardness (as CaCO3) of the test medium (untreated control) was determined to be 13°dH (232 mg/L CaCO3); the mean pH-value of the untreated control was determined to be 7.86 ± 0.05 (Std. Dev.), the mean temperature of the control and all test item concentrations was measured to be 20.2 ± 0.3 °C (Std. Dev.) and the mean oxygen concentration was determined to be 8.2 ± 0.8 mg/L (Std. Dev.).

Analytical Results:  The initial measured TOC content was between 71 and 95 % of nominal and the aged measured content was between 35 and 101 % of nominal. Since the TOC content in the initial samples was between 80 and 120 % of nominal the toxicological endpoints were evaluated using nominal concentrations.

Statistical Results: EC50 and NOEC-values of daphnids exposed to the test item evaluated using nominal concentrations

Findings:

Validity criteria: Control immobilisation: The percentage of immobilisation should be ≤ 10 %. In this study the control immobilisation was 0 %. Oxygen concentration: 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 ≥ 6.5 mg/L.

Test conditions: The total hardness (as CaCO3) of the test medium (untreated control) was determined to be 13°dH (232 mg/L CaCO3); the mean pH-value of the untreated control was determined to be 7.86 ± 0.05 (Std. Dev.), the mean temperature of the control and all test item concentrations was measured to be 20.2 ± 0.3 °C (Std. Dev.) and the mean oxygen concentration was determined to be 8.2 ± 0.8 mg/L (Std. Dev.).

Analytical Results: The initial measured TOC content was between 71 and 95 % of nominal and the aged measured content was between 35 and 101 % of nominal. Since the TOC content in the initial samples was between 80 and 120 % of nominal the toxicological endpoints were evaluated using nominal concentrations.

Statistical Results: EC50 and NOEC values of daphnids exposed to the test item evaluated using nominal concentrations:

24 h NOEC: 100 mg/L

48 h NOEC: 50.0 mg/L

24 h EC50 > 100 mg/L

48 h EC50 > 100 mg/L

Conclusion:

According to the results of the test, the EC50 (48 h) was proposed to be > 100 mg/L (nominal). The corresponding NOEC (48 h) was 50.0 mg/L (nominal).