Nitrile hydratase recombinant from E.coli

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 December 2020 to 14 January 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Remarks:

As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test material formulation.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: As the test material was a suspension and stored frozen, it was defrosted in a water bath at 25 °C for 65 minutes for the initial test and 55 minutes for the range-finding test and thoroughly mixed prior to weighing out. An initial test was initiated, however was terminated during being set-up due to the incorrect amount of test material being weighed out.
- In the range-finding test, activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg ai/L (three replicates for the test concentration of 1000 mg ai/L). The test material was dispersed directly in water.
- Nominal amounts of test material (32, 316 and 3165 mg (3165 mg in triplicate)) were each separately dispersed in approximately 200 mL of deionised reverse osmosis water prior to the measurement of the pH using a Hach HQ30d Flexi handheld meter and if necessary, adjusting to between pH 7.0 and 8.0. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg ai/L (three replicates at 1000 mg ai/L).

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate:
16 g Peptone
11 g Meat extract
3 g Urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
Each constituent was dissolved in a final volume of 1 L of water with the aid of ultrasonication.
The pH of the synthetic sewage stock used to feed the activated sewage sludge and used in the range-finding test was between pH 7.2 and 7.3. The pH values were measured using a Hach HQ30d Flexi handheld meter.

- Source: A mixed population of activated sewage sludge micro-organisms was obtained on 13 January 2021 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, UK which treats predominantly domestic sewage.
- The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC overnight prior to use in the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

measured temperature of between 19 and 21 °C

pH:

7.3 - 7.6

Nominal and measured concentrations:

10, 100 and 1000 mg ai/L

Details on test conditions:

TEST SYSTEM
- At time "0", 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air at a rate of 0.5 to 1.0 L per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item prior to the test material vessels being prepared. Finally, two further control vessels were prepared.
- Sludge concentration (weight of dry solids per volume): The suspended solids concentration was equal to 1.8 g/L and was adjusted to 3.0 g/L prior to the activated sewage sludge (5.5 L) being fed an aliquot of synthetic sewage (275 mL).

TEST WATER PARAMETERS
- The test water used for the test was deionis ed reverse osmosis water containing less than 1 mg/L Total Organic Carbon (TOC).

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the sample on the day of the test was 7.5 measured using a Hach HQ30d Flexi handheld meter.
- Photoperiod: The test was conducted under normal laboratory lighting in a temperature controlled room.

EFFECT PARAMETERS MEASURED
- Observations: Observations were made on the test preparations throughout the test period. Observations of the test item vessels at 0 hours were made prior to addition of activated sewage sludge.
- pH measurements: The pH of test preparations was measured at the test start (i.e. after the addition of activated sludge) and at the end of the 3-Hour incubation period using a Hach HQ40d Flexi handheld meter.
- Oxygen concentration: The oxygen concentrations in all vessels were measured after 30 minutes contact time.
- Measurement of the respiration rates: As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between 2 mg O2/L and 7 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.

TEST CONCENTRATIONS
- 10, 100 and 1000 mg ai/L (three replicates for the test concentration of 1000 mg ai/L).

DATA EVALUATION
- Calculation of the Oxygen Uptake Rates:
The respiration rate, R, expressed in milligrams oxygen per liter per hour (mg O2/L/h), was calculated from the linear part of the recorded oxygen decrease graph according to the following equation:

R = [(Q1 - Q2) / Δt] x 60

Where:
Q1 = The oxygen concentration at the beginning of the selected section of the linear phase (mg/L)
Q2 = The oxygen concentration at the end of the selected section of the linear phase (mg/L)
Δt = The time interval between the beginning and end of the selected section of the linear phase (minute).

The specific respiration rate, RS, expressed as the amount of oxygen consumed per gram dry weight of sludge per hour (mg O2/g/h) was deduced according to the following equation:

RS = R / SS

Where:
SS = The concentration of suspended solids in the test mixture (g dry weight/L).

- Calculation of Percentage of Inhibition:
The percentage inhibition was calculated according to the following equation:

Percentage inhibition = [1 – (R/Rbc)] x 100

Where:
Rbc = The mean respiration rate of the blank controls.

- ECX and No Observed Effect Concentration:
The percentage inhibition values were plotted against concentration for the reference item only, and the 3-Hour EC50 value was calculated by Probit analysis using linear maximum likelihood regression using the ToxRat computer software package (ToxRat® Solutions GMBH).
In order to determine the NOEC, Statistical Analysis using Williams Multiple Sequential t-test procedure incorporating Trend Analysis by contrasts, Shapiro-Wilk’s test on normal distribution and Levenes test on variance homogeneity procedure was carried out on the oxygen consumption data from the range-finding test to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the ToxRatPro software package.

An estimate of the 3-Hour ECx values for the test material were given by inspection of the inhibition data.

VALIDATION CRITERIA
The results of the study are considered valid if:
• The EC50 (3-Hour contact time) for 3,5-dichlorophenol lies within the range 2 to 25 mg/L for total respiration
• The specific respiration rate of the blank controls is not less than 20 mg oxygen per gram dry weight of sludge per hour for total respiration
• The coefficient of variation of oxygen uptake rate in control replicates is not more than 30 % at the end of the test

Reference substance (positive control):

yes

Remarks:

A reference item, 3,5-dichlorophenol, was included in the range-finding test at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum.

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 other: mg ai/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: inhibition of respiration rate

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 other: mg ai/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: inhibition of respiration rate

Details on results:

RANGE-FINDING TEST
- The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 % of the dissolved oxygen saturation level of 8.9 mg O2/L.
- No statistically significant toxic effects were shown at any of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.
- It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg ai/L.

VALIDATION CRITERIA
• The EC50 value (3-Hour contact time) for the reference item, 3,5-dichlorophenol, was 6.1 mg/L (recommended: within the range of 2 to 25 mg/L).
• The specific respiration rate of the controls was 32.09 mg oxygen per gram dry weight of sludge per hour (recommended: greater than 20 mg O2/g dry weight of sludge/hour).
• The coefficient of variation of oxygen uptake in the control vessels was 5.9 % at the end of the test (recommended: equal to or less than 30 %).
All validation criteria for the study were therefore satisfied.

INHIBITION OF RESPIRATION RATE
In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7 mg O2/L and 2 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

Results with reference substance (positive control):

INHIBITION OF RESPIRATION RATE
It was not possible to calculate an EC10 value for the reference item as the data generated was not suitable for the calculation method used to calculate this value.

Reported statistics and error estimates:

INHIBITION OF RESPIRATION RATE
See table 'Inhibition of Respiration Rate'.

Inhibition of Respiration Rate

	Test Material		Reference Material
	ECx (3 Hours) (mg ai/L)	95% Confidence Limits (mg/L)	ECx (3 Hours) (mg ai/L)	95% Confidence Limits (mg/L)
EC10	>1000	-	-	-
EC20	>1000	-	1.8	-
EC50	>1000	-	6.1	*
NOEC	1000	-	-	-

* It was not possible to calculate 95% confidence limits for the reference item due to insufficient number of replicates vessels to enable this calculation.
- = Not applicable

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 3 hour EC50 of the test material on activated sewage sludge micro-organisms was >1000 mg ai/L. The 3 hour NOEC was 1000 mg ai/L.

Executive summary:

The effect of the test material on the respiration of activated sewage sludge was investigated in accordance with the standardised guideline OECD 209, under GLP conditions. 

Activated sewage sludge was exposed to an aqueous dispersion of the test material at concentrations of 10, 100 and 1000 mg active ingredient (ai)/L (three replicates of the 1000 mg ai/L test concentration) for a period of 3 hours at a measured temperature of between 19 and 21 °C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg ai/L.

The reference item, 3,5-dichlorophenol, gave a 3-Hour EC50 value of 6.1 mg/L thereby confirming the acceptability of the inoculum used in the test system. It was not possible to calculate 95 % confidence limits for the reference item due to insufficient number of replicates vessels to enable this calculation. 

Under the conditions of this study, the 3 hour EC50 of the test material on activated sewage sludge micro-organisms was >1000 mg ai/L. The 3 hour NOEC was 1000 mg ai/L.

Description of key information

Under the conditions of this study, the 3 hour EC50 of the test material on activated sewage sludge micro-organisms was >1000 mg ai/L. The 3 hour NOEC was 1000 mg ai/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

The effect of the test material on the respiration of activated sewage sludge was investigated in accordance with the standardised guideline OECD 209, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997). 

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg active ingredient (ai)/L (three replicates of the 1000 mg ai/L test concentration) for a period of 3 hours at a measured temperature of between 19 and 21 °C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg ai/L.

The reference item, 3,5-dichlorophenol, gave a 3-Hour EC50 value of 6.1 mg/L thereby confirming the acceptability of the inoculum used in the test system. It was not possible to calculate 95 % confidence limits for the reference item due to insufficient number of replicates vessels to enable this calculation. 

Under the conditions of this study, the 3 hour EC50 of the test material on activated sewage sludge micro-organisms was >1000 mg ai/L. The 3 hour NOEC was 1000 mg ai/L.