Nitrile hydratase recombinant from E.coli

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 January 2021 to 14 February 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Before use, the test item was defrosted in a water bath at 25 ºC 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 test preparation prior to inoculation at 0 hours and from additional media containing no algal cells ran alongside the test at 72 hours. 0-Hour samples were stored refrigerated prior to TOC analysis alongside the 72-Hour samples at the end of the test. A set of duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Range-Finding Test Method: A nominal amount of test material (633 mg) was dispersed in culture medium and the volume adjusted to 1 L to give a 100 mg a.i./L stock dispersion from which a series of dilutions was made to give further stock solutions of 1.0 and 10 mg a.i./L. An aliquot (500 mL) of each of the stock solutions was separately inoculated with algal suspension (2.5 mL) to give an initial nominal cell density of 5.00 x 10^3 cells/mL.
The stock dispersion and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

- Definitive Test Method: A nominal amount of test material (1266 mg) was dispersed in culture medium and the volume adjusted to 2 L to give a 100 mg a.i./L stock solution from which a series of dilutions was made to give further stock dispersions of 1.0, 3.2, 10 and 32 mg a.i./L. An aliquot (500 mL) of each of the stock solutions was separately inoculated with algal suspension (1.8 mL) to give an initial nominal cell density of 5.00 x 10^3 cells/mL.
The stock dispersions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

- Other relevant information: At the start of the test, all control, 1.0 and 3.2 mg a.i./L test cultures were observed to be clear colorless solutions. The 10 mg a.i./L test cultures were observed to be very slightly off-white cloudy dispersions, the 32 mg a.i./L test cultures were observed to be slightly cloudy off-white dispersions and the 100 mg a.i./L test cultures were observed to be off-white cloudy dispersions. After the 72-Hour test period all control, 1.0, 3.2, 10 and 32 mg a.i./L test cultures were observed to be green dispersions whilst the 100 mg a.i./L test cultures were observed to be green cloudy dispersions.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name:
- Strain: CCAP 278/4

ACCLIMATION
- Not specified

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C

pH:

7.8 - 9.4

Nominal and measured concentrations:

Definitive test: 1.0, 3.2, 10, 32 and 100 mg a.i./L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type: Plugged with polyurethane foam bungs to reduce evaporation
- Fill volume: 100 mL of test preparation
- Aeration: The flasks were kept under constant agitation by orbital shaker (approximately 150 rpm)
- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 1.40 x 10^6 cells per mL. Inoculation of 500 mL of test medium with 1.8 mL of this algal suspension gave an initial nominal cell density of 5.00 x 1^03 cells per mL and had no significant dilution effect on the final test concentration.
- Control end cells density: Approximately 10^5 to 10^6 cells/mL
- No. of vessels per concentration (replicates): Three flasks each containing 100 mL were used for each treatment group
- No. of vessels per control (replicates): Six flasks each containing 100 mL of test preparation were used for the control

CULTURE MEDIUM
- Standard medium used: not specified. The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+ or Elga Purelab Option R-15 BP) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
- Detailed composition if non-standard medium was used:
NaNO3, 25.5 mg/L
MgCl2.6H2O, 12.16 mg/L
CaCl2.2H2O, 4.41 mg/L
MgSO4.7H2O, 14.6 mg/L
K2HPO4, 1.044 mg/L
NaHCO3, 15.0 mg/L
H3BO3, 0.186 mg/L
MnCl2.4H2O, 0.415 mg/L
ZnCl2, 0.00327 mg/L
FeCl3.6H2O, 0.160 mg/L
CoCl2.6H2O, 0.00143 mg/L
Na2MoO4.2H2O, 0.00726 mg/L
CuCl2.2H2O, 0.000012 mg/L
Na2EDTA.2H2O, 0.30 mg/L
- Culture medium same as growth medium: Yes. The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: intensity approximately 7000 lux provided by warm white lighting (380 to 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Test organism observations:
Samples were taken at 22, 48 and 72 hours and the cell densities determined using a haemocytometer and light microscope. Three determinations were made for each sample. The nominal inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

TEST CONCENTRATIONS
Range finding study
- Test concentrations: Nominal test concentrations of 1.0, 10 and 100 mg active ingredient (a.i.)/L
- Results used to determine the conditions for the definitive study: Yes. Based on the results of the range-finding test (the results showed no effect on growth at the test concentration of 1.0 mg a.i./L. However, growth and yield were observed to be reduced at 10 and 100 mg a.i./L.) the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg a.i./L. The amount of test material used was corrected for a purity value of 15.8%.

DATA EVALUATION
- Comparison of Growth Rates
The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from the following equation:

μ = lnNn - lnN1 / tn - t1

Where
μ = average specific growth rate from time t1 to tn
N1 = cell concentration at t1
Nn = cell concentration at tn
t1 = time of first measurement
tn = time of nth measurement

The average specific growth rate over the test duration was calculated for each replicate control and test item vessel using the nominal inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision of the calculation.

In addition the section by section specific growth rate (Days 0 to 1, 1 to 2 and 2 to 3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant.

Percentage inhibition of growth rate for each replicate test item vessel was calculated using the following equation:

Ir = (μc - μt / μc) x 100

Where:
Ir = percentage inhibition of average specific growth rate
μc = mean average specific growth rate for the control cultures
μt = average specific growth rate for the test culture

- Comparison of Yield
Yield is calculated as the increase in biomass over the exposure period using the following equation:

Y = Nn - N0

Where:
Y = yield
N0 = cell concentration at the start of the test
Nn = cell concentration at the end of the test

For each test concentration and control the mean value for yield along with the standard deviation was calculated. Percentage inhibition of yield was calculated using the following equation:

Iy = (Yc - Yt / Yc) x 100

Where:
Iy = percentage inhibition of yield
Yc = mean value for yield in the control group
Yt = mean value for yield for the treatment group

- Determination of ECx Values
All ECx values and associated confidence limits and the slope of the response curve and its standard error for growth rate and yield were calculated by Probit analysis using Linear Maximum-Likelihood regression.

All ECx values were determined using the ToxRat computer software package (ToxRat® Solutions GMBH).

- Lowest Observed Effect Concentration and No Observed Effect Concentration Determinations
Prior to determination of the Lowest Observed Effect Concentration (LOEC) and No Observed Effect Concentration (NOEC), data for each endpoint was assessed using Shapiro-Wilk’s test on Normal Distribution, Levene’s test on Variance Homogeneity and Non-parametric Trend analysis by contrasts to enable the correct comparison test to be performed. The Step-down Jonckheere-Terpstra test procedure was used for both growth rate and yield.

All statistical analyses were performed using the ToxRat computer software package (ToxRat® Solutions GMBH).

VALIDATION CRITERIA
The results of the test are considered valid if the following performance criteria are met:
• The cell concentration of the control cultures must increase by a factor of at least 16 over the test period
• The mean of the coefficients of variation of the section by section daily growth rates in the control cultures during the course of the test (Days 0 to 1, 1 to 2 and 2 to 3, for the 72-Hour tests) must not exceed 35 %
• The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7 %

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:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

yield

Details on results:

GROWTH DATA
- From the data given in Table 'Cell Densities and pH Values in the Definitive Test' and Table 'Inhibition of Growth Rate and Yield in the Definitive Test', it is clear that the growth rate (r) and yield (y) of Raphidocelis subcapitata (CCAP 278/4) were affected by the presence of the test material over the 72-Hour exposure period.

VALIDITY CRITERIA
The following data show that the cell concentration of the control cultures increased by a factor of 171 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Nominal cell density of control at 0 hours : 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 8.56 x 10^5 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 12 % and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35 %.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hour) was 6% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7 %.

OBSERVATIONS ON CULTURES
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 1.0 and 3.2 mg a.i./L, however some enlarged cells were observed to be present in the test cultures at 10 and 32 mg a.i./L and misshapen cells and cell debris were observed to be present in the test cultures at 100 mg a.i./L.

Results with reference substance (positive control):

Exposure of Raphidocelis subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour): 1.3 mg/L; 95 % confidence limits 1.2 to 1.5 mg/L
EyC50 (0 to 72 hour): 0.44 mg/L; 95 % confidence limits 0.37 to 0.52 mg/L
No Observed Effect Concentration based on growth rate: 0.125 mg/L
No Observed Effect Concentration based on yield: 0.125 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.25 mg/L
Lowest Observed Effect Concentration based on yield: 0.25 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item (In-house data from the previous five studies shows a mean ErC50 value of 1.4 mg/L (standard deviation = 0.16) and a mean EyC50 value of 0.64 mg/L (standard deviation = 0.12).

Reported statistics and error estimates:

INHIBITION OF GROWTH RATE
ErC10 (0 to 72 hour): 99 mg a.i./L; 95 % confidence limits 36 to 1368 mg a.i./L
ErC20 (0 to 72 hour): >100 mg a.i./L
ErC50 (0 to 72 hour): >100 mg a.i./L

where ErCx is the test concentration that reduced growth rate by x %.

Statistical analysis of the growth rate data was carried out for the control and all test concentrations using Step-down Jonckheere-Terpstra test procedure incorporating Shapiro-Wilk’s test on Normal Distribution, Levene’s test on Variance Homogeneity and Non-parametric Trend analysis by contrasts. There were no statistically significant differences (P≥0.05), between the control and 1.0, 3.2, 10 and 32 mg a.i./L test concentrations; however, the 100 mg a.i./L test concentration was significantly different (P<0.05) and, therefore the NOEC based on growth rate was 32 mg a.i./L. Correspondingly the Lowest Observed Effect Concentration (LOEC) based on growth rate was 100 mg a.i./L.

INHIBITION OF YIELD
EyC10 (0 to 72 hour): 9.7 mg a.i./L; 95% confidence limits 0.42 to 226 mg a.i./L
EyC20 (0 to 72 hour): 23 mg a.i./L; 95% confidence limits 3.1 to 166 mg a.i./L
EyC50 (0 to 72 hour): >100 mg a.i./L

Where EyCx is the test concentration that reduced yield by x %.

Statistical analysis of the yield data was carried out as for inibition of growth rate. There were no statistically significant differences (P≥0.05), between the control and 1.0, 3.2, 10 and 32 mg a.i./L test concentrations; however, the 100 mg a.i./L test concentration was significantly different (P<0.05) and, therefore the NOEC based on yield was 32 mg a.i./L. Correspondingly the LOEC based on yield was 100 mg a.i./L.

Cell Densities and pH Values in the Definitive Test

Nominal Concentration		pH	Dell densities* (cells per mL)			pH
		0 hours	22 hours	48 hours	72 hours	72 hours
Control	R1	7.8	2.34E+04	2.78E+0.5	1.37E+06	9.4
	R2		2.17E+04	1.20E+05	6.90E+05
	R3		1.84E+04	1.43E+05	6.80E+05
	R4		2.67E+04	1.45E+05	8.57E+05
	R5		2.34E+04	1.58E+05	7.97E+05
	R6		3.00E+04	1.22E+05	7.43E+05
	Mean		2.39E+04	1.61E+05	8.56E+05
1.0	R1	7.8	2.67E+04	1.30E+05	6.43E+05	9.2
	R2		4.50E+04	1.25E+05	7.17E+05
	R3		2.00E+04	1.48E+05	7.63E+05
	Mean		3.06E+04	1.34E+05	7.08E+05
3.2	R1	7.7	2.34E+04	9.17E+04	9.40E+05	9.3
	R2		2.84E+04	1.00E+05	6.30E+05
	R3		1.50E+04	1.27E+05	6.30E+05
	Mean		2.23E+04	1.06E+05	7.33E+05
10	R1	7.7	2.34E+04	1.17E+05	8.63E+05	9.2
	R2		1.67E+04	1.20E+05	8.47E+05
	R3		1.84E+04	1.15E+05	8.63E+05
	Mean		1.95E+04	1.17E+05	8.58E+05
32	R1	7.6	6.65E+03	1.07E+05	6.00E+05	9.3
	R2		5.00E+03	1.25E+05	6.67E+05
	R3		5.00E+03	1.32E+05	6.57E+05
	Mean		5.55E+03	1.21E+05	6.41E+05
100	R1	7.6	3.35E+03	7.67E+04	4.47E+05	8.6
	R2		1.50E+04	8.50E+04	4.70E+05
	R3		6.65E+03	6.84E+04	4.03E+05
	Mean		8.33E+03	7.67E+04	4.40E+05

*Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 fields of view for each of the replicate flasks
R = Replicate

 

 

Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (mg a.i./L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.078	-	1.37E+06	-
	R2	0.068		6.85E+05
	R3	0.068		6.75E+05
	R4	0.071		8.52E+05
	R5	0.070		7.92E+05
	R6	0.069		7.38E+05
	Mean	0.071		8.51E+05
	SD	0.004		2.60E+05
1.0	R1	0.067	6	6.38E+05
	R2	0.069	3	7.12E+05
	R3	0.070	1	7.58E+05
	Mean	0.069	3	7.03E+05	17
	SD	0.002		6.05E+04
3.2	R1	0.073	[3]	9.35E+05
	R2	0.067	6	6.25E+05
	R3	0.067	6	6.25E+05
	Mean	0.069	3	7.28E+05	14
	SD	0.003		1.79E+05
10	R1	0.072	[1]	8.58E+05
	R2	0.071	0	8.42E+05
	R3	0.072	[1]	8.58E+05
	Mean	0.072	[1]	8.53E+05	0
	SD	0.001		9.24E+03
32	R1	0.066	7	5.95E+05
	R2	0.068	4	6.62E+05
	R3	0.068	4	6.52E+05
	Mean	0.067	5	6.36E+05	25
	SD	0.001		3.61E+04
100	R1	0.062	13	4.42E+05
	R2	0.063	11	4.65E+05
	R3	0.061	14	3.98E+05
	Mean	0.062	13	4.35E+05	49
	SD	0.001		3.40E+04

*In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated
R = Replicate
SD = Standard Deviation
[ ] = Increase in growth compared to controls
- = Not applicable

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study the EC50 based on growth rate and yield was determined to be > 100 mg a.i/L. The NOEC based on growth rate and yield was determined to be 32 mg a.i/L. The LOEC based on growth rate and yield was determined to be 100 mg a.i/L. (95% confidence limits not determined). 

Executive summary:

The effect of the test material on the growth of the green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) was investigated in accordance with the standardised guidelines OECD 201 and EU Method C.3, under GLP conditions. 

Following a preliminary range-finding test, Raphidocelis subcapitata was exposed to aqueous dispersions of the test material at concentrations of 1.0, 3.2, 10, 32 and 100 mg active ingredient (a.i.)/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a haemocytometer and light microscope.

Total Organic Carbon (TOC) analysis of the test preparations at 0 and 72 hours showed measured carbon concentrations to range from less than the Limit of Quantification (LOQ) equivalent to 1.0 mg C/L to 41.46 mg C/L and less than the LOQ to 22.50 mg C/L respectively.

Under the conditions of this study the EC50 based on growth rate and yield was determined to be > 100 mg ai.i/L. The NOEC based on growth rate and yield was determined to be 32 mg a.i/L. The LOEC based on growth rate and yield was determined to be 100 mg a.i/L. (95% confidence limits not determined). 

Description of key information

Under the conditions of this study the EC50 based on growth rate and yield was determined to be > 100 mg ai.i/L. The NOEC based on growth rate and yield was determined to be 32 mg a.i/L. The LOEC based on growth rate and yield was determined to be 100 mg a.i/L. (95% confidence limits not determined). 

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

32 mg/L

Additional information

The effect of the test material on the growth of the green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) was investigated in accordance with standardised guidelines OECD 201 and EU Method C.3, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997). 

Following a preliminary range-finding test, Raphidocelis subcapitata was exposed to aqueous dispersions of the test item at concentrations of 1.0, 3.2, 10, 32 and 100 mg active ingredient (a.i.)/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a haemocytometer and light microscope.

Total Organic Carbon (TOC) analysis of the test preparations at 0 and 72 hours showed measured carbon concentrations to range from less than the Limit of Quantification (LOQ) equivalent to 1.0 mg C/L to 41.46 mg C/L and less than the LOQ to 22.50 mg C/L respectively.

Under the conditions of this study the EC50 based on growth rate and yield was determined to be > 100 mg ai.i/L. The NOEC based on growth rate and yield was determined to be 32 mg a.i/L. The LOEC based on growth rate and yield was determined to be 100 mg a.i/L. (95% confidence limits not determined).