Nitrile hydratase recombinant from E.coli

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:

21 January 2021 to 21 April 2021

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)

Version / remarks:

April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The amount of test material used was corrected for a purity value of 15.8%.
Before use, the test material was defrosted in a water bath at 25 °C for 90 minutes and shaken several times.

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk fresh test preparations at 0 and 24 hours and from the pooled replicates at 24 and 48 hours from the old media for immediate analysis. Duplicate sets of samples were taken at 0, 24 and 48 hours and stored frozen for further analysis if necessary.

Vehicle:

yes

Remarks:

test water

Details on test solutions:

DEFINITIVE TEST
A nominal amount of test material (1266 mg) was dissolved in test water and the volume adjusted to 2 L to give the 100 mg a.i./L test concentration from which a series of dilutions was made to give further test concentrations of 10, 18, 32 and 56 mg a.i./L.
Each stock dispersion and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Total Organic Carbon analysis was performed on the test dispersions at 0, 24 and 48 hours

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Species: Daphnia magna
- Age at study initiation: Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. The young were removed from the cultures and used for testing.
- Source: The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures
- Method of breeding: Culture conditions ensured that reproduction was by parthenogenesis
- Feeding: Each culture was fed daily with a mixture of algal suspension (Raphidocelis subcapitata) and GEMMA Micro 300 fish food suspension. The young daphnids used for the test received no food during exposure.
- Culture conditions: Adult daphnids were maintained in 150 mL glass vessels containing 100 mL Elendt M4 medium in a temperature controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

2.5 mmol/L

Test temperature:

20 °C

pH:

7.3 - 8.2

Dissolved oxygen:

1.0 to 9.0 mg O2/L

Nominal and measured concentrations:

Nominal concentrations of 10, 18, 32, 56 and 100 mg active ingredient (a.i.)/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass vessels containing approximately 100 mL of test preparation were used
- Type: closed (the test vessels were covered to reduce evaporation)
- Aeration: The contents of the test vessels were not aerated.
- Renewal rate of test solution: Test media renewal at 24 hours (the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media).
- No. of organisms per vessel: At the start of the test, five daphnids were placed in each test and control vessel at random containing the test preparations
- No. of vessels per concentration and control (replicates): Four replicate test and control vessels were prepared.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (Elendt M4 medium) used for the range-finding and definitive tests. The composition of the Reconstituted water (Elendt M4 medium) was as follows:
CaCl2.2H2O: 293.8 mg/L
MgSO4.7 H2O: 123.3 mg/L
NaHCO3: 64.8 mg/L
KCl: 5.8 mg/L
Na2SiO3.9 H2O: 10.0 mg/L
NaNO3: 0.274 mg/L
K2HPO4: 0.184 mg/L
KH2PO4: 0.143 mg/L
H3BO3: 2.8595 mg/L
Na2EDTA.2 H2O: 2.50 mg/L
FeSO4.7 H2O: 0.9955 mg/L
MnCl2.4H2O: 0.3605 mg/L
LiCl 0.306: mg/L
SrCl2.6 H2O: 0.152 mg/L
RbCl 0.071: mg/L
Na2MoO4.2 H2O: 0.0615 mg/L
CuCl2.2 H2O: 0.0165 mg/L
NaBr 0.0160: mg/L
ZnCl2 0.0130: mg/L
CoCl2.6 H2O: 0.010 mg/L
KI: 0.00325 mg/L
Na2SeO3: 0.00219 mg/L
NH4VO3: 0.000575 mg/L
Thiamine hydrochloride: 0.075 μg/L
Cyanocobalamine (Vitamin B12): 0.0010 μg/L
D(+) biotin (Vitamin H): 0.00075 μg/L
- Water Hardness: 2.5 mmol/L (= 250 mg/L as CaCO3)
- Alkalinity: 0.9 mmol/L
- The pH of the prepared media was in the range of 7.9 ± 0.3 and stored at approximately 21 °C. If necessary the pH was adjusted with NaOH or HCl.

OTHER TEST CONDITIONS
- Photoperiod: photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods
- Light intensity: between 200 and 1200 Lux

EFFECT PARAMETERS MEASURED:
- Test Organism Observations: Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim within 15 seconds after gentle agitation.
As the test item formed a dispersion, observations were conducted on any immobilised daphnia to identify any encapsulation or entrapment.
- Water Quality Criteria:
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24 hour old test preparations. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a digital thermometer.
The light intensity during the light period was measured using an ATP Instrumentation Lux meter.
The appearance of the test media was recorded daily.

CONTROL PERFORMED: yes, the control group was maintained under identical conditions but not exposed to the test material.

RANGE-FINDING STUDY
- Test concentrations: 1.0, 10 and 100 mg a.i./L
- Results used to determine the conditions for the definitive study: yes (based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 mg a.i./L)

Reference substance (positive control):

yes

Remarks:

A positive control test using potassium dichromate as the reference item was performed twice in a 12 month period to demonstrate satisfactory conditions of the test.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

RANGE-FINDING TEST
No sub-lethal effects of exposure were observed throughout the test.
No immobilisation was observed at the test concentrations of 1.0 and 10 mg a.i./L, however; 50% immobilisation was observed at 100 mg a.i./L.
Based on this information test concentrations of 10, 18, 32, 56 and 100 mg a.i./L were selected for the definitive test.
Media used for the range-finding test was found to have a hardness below the acceptable limits (250 mg/L as CaCO3 ± 20 mg/L as CaCO3) at 226 mg/L as CaCO3. As no immobilisation or sub lethal effects were observed in the control, the reduced hardness was taken to have had no adverse effect on the test. Oxygen concentration was recorded to have dropped to 1.93 mg O2/L at 48 hours at 100 mg a.i./L. As the validity criteria was not applied in the range-finding test this was not considered to have had an adverse effect on the test.
TOC analysis of the test preparations at 0 hours showed measured carbon concentrations to range from 0.43 to 50.17 mg C/L. At 48 hours, measured carbon concentrations ranged from less than the control to 11.12 mg C/L.

DEFINITIVE TEST
VERIFICATION OF TEST CONCENTRATIONS
Total Organic Carbon (TOC) analysis (see table 'Total Organic Carbon Analysis - Definitive test results') of the fresh test preparations at 0 and 24 hours showed measured carbon concentrations to range from 4.65 to 47.85 mg C/L and 4.02 to 43.03 mg C/L respectively. In the old test preparations at 24 and 48 hours, results showed measured carbon concentrations to range from 2.16 to 31.83 mg C/L and 2.28 to 24.72 mg C/L respectively. Given that TOC may have been one or several components of the test material, the toxicity cannot be attributed to a single component or a mixture of components, but to the test material as a whole, the results were based on nominal loading rates only.

IMMOBILISATION DATA
Cumulative immobilisation data and other observations from the exposure of Daphnia magna to the test material during the definitive test are given in Table 'Cumulative Immobilisation Data and Observations in the Definitive Test'.
Inspection of the data at 24 hours and analysis of the immobilisation data by Probit analysis using Linear Maximum-Likelihood regression at 48 hours based on the nominal test concentrations gave the results shown in table 'Results'.
No Observed Effect Concentration after 24 and 48 hours exposure was 100 mg a.i./L.
The slope and standard error of the response curve at 48 hours was 1.8 (standard error = 0.33). Due to the nature of the data it was not possible to calculate the slope and error of response curve at 24 hours.
It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg a.i./L.
Microscopic observations conducted on all immobilised daphnids confirmed no precipitate to be visible adhered to the daphnia.

SUB-LETHAL EFFECTS
No sub-lethal effects of exposure were observed throughout the test (see Table 'Cumulative Immobilisation Data and Observations in the Definitive Test').

WATER QUALITY CRITERIA
Temperature was maintained at approximately 20 °C throughout the test. While there were no treatment related differences for pH, concentration dependent differences in oxygen concentration were observed throughout the test.
Throughout the test the light intensity was observed to be in the range 816 to 831 Lux.

OBSERVATIONS ON TEST ITEM SOLUBILITY
At the start and throughout the test all control solutions were observed to be clear colourless solutions. The fresh test preparations were observed to be slightly hazy dispersions, hazy dispersions, very hazy off white dispersions, cloudy off white dispersions and very cloudy off white dispersions at 10, 18, 32, 56 and 100 mg a.i./L respectively. The old test preparations were observed to be very slightly hazy dispersions, slightly hazy dispersions, hazy dispersions, slightly cloudy off white dispersions and cloudy off white dispersions at 10, 18, 32, 56 and 100 mg a.i./L respectively.


VALIDATION CRITERIA
The results of the test are considered valid if the following performance criteria are met:
- No more than 10% of the control daphnids show immobilisation or other signs of disease or stress (e.g. discoloration or unusual behaviour such as trapping at the surface water).
- The dissolved oxygen concentration at the end of the test is equal to or greater than 3 mg/L in the control and test vessels.

The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress. The oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control however, a concentration dependent decrease was observed in all old dispersions. As this appears to be an effect of the test material and therefore unavoidable, the test is considered to be valid.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. The positive control was conducted between 05 January 2021 and 29 January 2021.
Analysis of the immobilisation data was carried out using the Weibull analysis using linear maximum-likelihood regression at 24 and 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package. Accordingly the following results were obtained (See table 'Positive control results').
The NOEC is based upon equal to or less than 10% immobilisation at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item*.

Reported statistics and error estimates:

An estimate of the 24-Hour EC50 value was given by inspection of the immobilisation data. The EC50 value and associated confidence limits at 48 hours and the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum-Likelihood regression. The Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) at 24 and 48 hours were calculated using the Qualitative Trend Analysis by Contrasts prior to Fisher’s Exact Binomial Test with Bonferroni correction. All results were calculated using the ToxRat computer software package (ToxRat® Solutions GMBH).

Total Organic Carbon Analysis - Definitive test results

Samples	Nominal Loading Rate (mg a.i./L)	Concentration of TOC (mg C/L)	Concentration of TOC Corrected for Control (mg C/L)
0 Hours	Control	1.12	-
	10	5.77	4.65
	18	9.80	8.68
	32	16.26	15.14
	56	27.96	26.84
	100	48.97	47.85
24 Hours (Old Media)	Control	1.70	-
	10	3.86	2.16
	18	6.15	4.45
	32	10.23	8.53
	56	17.83	16.13
	100	33.53	31.83
24 Hours (Fresh Media)	Control	1.74	-
	10	5.76	4.02
	18	9.04	7.30
	32	14.63	12.89
	56	24.91	23.17
	100	44.77	43.03
48 Hours	Control	1.04	-
	10	3.32	2.28
	18	5.41	4.37
	32	9.26	8.22
	56	14.94	13.90
	100	25.76	24.72

 

Cumulative Immobilisation Data and Observations in the Definitive Test

Nominal Concentration (mg a.i./L)	24 Hours
	Cumulative Immobilised Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
10	0	0	0	0	0	0	5 N	5 N	5 N	5 N
18	0	0	0	0	0	0	5 N	5 N	5 N	5 N
32	0	0	0	0	0	0	5 N	5 N	5 N	5 N
56	0	0	0	0	0	0	5 N	5 N	5 N	5 N
100	0	0	0	0	0	0	5 N	5 N	5 N	5 N
Nominal Concentration (mg a.i./L)	48 Hours
	Cumulative Immobilised Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
10	0	0	0	0	0	0	5 N	5 N	5 N	5 N
18	0	0	0	0	0	0	5 N	5 N	5 N	5 N
32	0	0	0	1	1	5	5 N	5 N	5 N	4 N
56	1	0	0	2	3	15	4 N	5 N	5 N	3 N
100	1	0	1	1	3	15	4 N	5 N	4 N	4 N

 

R           = Replicate

N           = Normal

 

Results

Time (Hour)	EC50 (mg a.i./L)	95% Confidence limits (mg a.i./L)
24	>100	Not determined
48	>100	Not determined

 

Positive control results

Time Point (Hours)	EC50 (mg/L)	95% Confidence Limits (mg/L)			NOEC (mg/L)	LOEC (mg/L)
24	0.86	0.21	-	3.5	0.32	0.56
48	0.50	0.43	-	0.58	0.32	0.56

 

NOEC = No observed effect concentration

LOEC = Lowest observed effect concentration

*The results of the inter laboratory tests and a Technical Corrigendum to ISO 6341 give a 24-Hour EC50 for potassium dichromate within the range 0.6 mg/L to 2.1 mg/L

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the acute toxicity of the test material to the freshwater invertebrate Daphnia magna gave a 48-Hour EC50 value of greater than 100 mg a.i./L and a NOEC of 100 mg a.i./L.

Executive summary:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna was investigated under GLP conditions and according to OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test"  and Method C.2 of Commission Regulation (EC) No. 440/2008.

Following a preliminary range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to aqueous dispersions of the test item at nominal concentrations of 10, 18, 32, 56 and 100 mg active ingredient (a.i.)/L for 48 hours at a temperature of approximately 20 °C under semi-static test conditions. The number of immobilized Daphnia was recorded after 24 and 48 hours.

Total Organic Carbon (TOC) analysis of the fresh test preparations at 0 and 24 hours showed measured carbon concentrations to range from 4.65 to 47.85 mg C/L and 4.02 to 43.03 mg C/L respectively. In the old test preparations at 24 and 48 hours, results showed measured carbon concentrations to range from 2.16 to 31.83 mg C/L and 2.28 to 24.72 mg C/L respectively. Given that TOC may have been one or several components of the test item, the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg a.i./L.

Under the conditions of this study, the acute toxicity of the test material to the freshwater invertebrate Daphnia magna gave a 48-Hour EC50 value of greater than 100 mg a.i./L and a NOEC of 100 mg a.i./L.

Description of key information

The short-term toxicity of the test material to the freshwater invertebrate Daphnia magna gave a 48-Hour EC50 value of greater than 100 mg a.i./L and a NOEC of 100 mg a.i./L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

> 100 mg/L

Additional information

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna was investigated under GLP conditions and according to OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test"  and Method C.2 of Commission Regulation (EC) No. 440/2008.

Following a preliminary range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to aqueous dispersions of the test item at nominal concentrations of 10, 18, 32, 56 and 100 mg active ingredient (a.i.)/L for 48 hours at a temperature of approximately 20 °C under semi-static test conditions. The number of immobilized Daphnia was recorded after 24 and 48 hours.

Total Organic Carbon (TOC) analysis of the fresh test preparations at 0 and 24 hours showed measured carbon concentrations to range from 4.65 to 47.85 mg C/L and 4.02 to 43.03 mg C/L respectively. In the old test preparations at 24 and 48 hours, results showed measured carbon concentrations to range from 2.16 to 31.83 mg C/L and 2.28 to 24.72 mg C/L respectively. Given that TOC may have been one or several components of the test item, the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg a.i./L.

Under the conditions of this study, the acute toxicity of the test material to the freshwater invertebrate Daphnia magna gave a 48-Hour EC50 value of greater than 100 mg a.i./L and a NOEC of 100 mg a.i./L.