Escherichia coli dehalogenase catalyst

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2nd July 2018 to 1st 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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition 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 were taken at 0 hours (initial value) from fresh test solutions and after 72 hours from aged test solutions from all test item concentrations, and control. For each sampling also retain sample was taken. 20 mL samples were taken.
- Sample storage conditions before analysis: All samples were stored at room temperature until they were transferred to the analytical laboratory.

Test solutions

Vehicle:

no

Details on test solutions:

The following nominal test item concentrations were employed (spaced by a factor of 3.2): 100, 31.3, 9.77, 3.05 and 0.954 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 was added up to the bench mark and the solution was homogenised by shaking Afterwards the stock solution S1 was turbid. Lower test solutions were prepared by dilution of appropriate solutions with test medium. Dilution V1 was also turbid and dilutions V2 – V4 were clear and transparent. Algae were added to each solution individually targeting nominal cell densities of 0.5 × 104 cells per mL in each solution. The preparation of the test solutions is shown below.
One replicate per treatment without algae was run in parallel which was used for the analytical sampling.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata Hindák (Sphaeropleales: Selenastraceae)
- Strain: SAG 61.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany.
- Age of inoculum (at test initiation): Not specified
- Method of cultivation:

The algae are grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Stock cultures are ordered regularly from the commercial supplier.
Culture conditions are as follows:
• Illumination: continuously (approx. 4440 - 8880 lux at cell culture level or 60 - 120 µEm-2s-1)
• Temperature: 21 - 24 °C
• Culture flasks: 100 mL Erlenmeyer flasks
• CO2 supply by shaking on a rotating shaker, approximately 105 rpm
Cells from this semi-continuous liquid stock culture were used for the test.
3 to 4 days before start of the test, test medium was inoculated with the test organism and held under test conditions in order to produce a pre-culture in the state of exponential growth.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

Not specified

Test temperature:

22.5 – 23.3 °C

pH:

7.32 – 7.63

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal test item concentrations of 100, 31.3, 9.77, 3.05, 0.954 mg/L.
The measured initial TOC content ranged from 81 % to 108 % of nominal. In the aged samples the measured TOC content was between 80 % and 112 %. Therefore toxicological endpoints were evaluated using the nominal concentrations of the test item.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (100 mL) with aluminium caps were filled up with ~ 50 mL test solution.
- Aeration: Not specified
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- Initial cells density: 0.5 × 104 cells/mL.
- No. of vessels per concentration (replicates): Three replicates were employed for each of the test item concentrations.
- No. of vessels per control (replicates): Six replicates for the control.
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes. The medium used for the test was AAP-Medium.
- Detailed composition if non-standard medium was used: Not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Not specified
- Total organic carbon: Not specified
- Particulate matter: Not specified
- Metals: Not specified
- Pesticides: Not specified
- Chlorine: Not specified
- Alkalinity: Not specified
- Ca/mg ratio: Not specified
- Conductivity: Not specified
- Culture medium different from test medium: Not specified
- Intervals of water quality measurement: Not specified

OTHER TEST CONDITIONS
- Sterile test conditions: Not specified
- Adjustment of pH: The pH of the test medium was adjusted to 7.5 +/- 0.1 with NaOH and HCl; pH of control: 7.32 – 7.63.
- Photoperiod: Continuously
- Light intensity and quality: 88.1 µEm-2s-1 (mean)
- Salinity (for marine algae): Not applicable
The incubation took place in a temperature controlled light incubator. The incubator is equipped with a pivoted bogie consisting of several platforms. The bogie turns around passing laterally placed fluorescent tubes, which enables uniform lighting of the test vessels.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At 24, 48 and 72 hours from the test initiation, the number of cells in each replicate was determined by fluorescence detection and used to calculate effect concentrations for the test item, which resulted in 10, 20 and 50 % inhibition of the cell growth rate (ErC10, 20, 50) and yield (EyC10, 20, 50). The test item concentration which did not cause any significant inhibition (NOEC) and the lowest observed effect concentration (LOEC) were also determined where possible.
Additionally, the morphological appearance of the algae cells was observed microscopically at the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: Not applicable
- Test concentrations: 100, 31.3, 9.77, 3.05 and 0.954 mg/L and control
- Results used to determine the conditions for the definitive study: Not applicable

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 7

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The morphology of the algae cells was observed microscopically at test end. The cells were considered normal for the control and up to and including a nominal test item concentration of 100 mg/L.
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No
After 24 h and 48 h there were rose beige substances on the bottom of the test vessels at test item concentrations of 31.3 and 100 mg/L. The appearance of these test item solution changed into green flakes after 72h.- Exponential growth in the control (for algal test): yes
The morphology of the algae cells was observed microscopically at test end. The cells were considered normal for the control and up to and including a nominal test item concentration of 100 mg/L.
The growth conditions (pH and temperature) during the test were within the range specified by OECD 201.
The mean light intensity of all positions of the incubator was 88.1 µEm-2s-1 with mean values of each platform in a range from 82.9 to 92.5 µEm-2s-1 (-5.9 % to +5.0 % of mean) which was within ± 15 % of variation as specified by OECD 201

Results with reference substance (positive control):

The ErC10 value for growth rate was determined to be 0.863 mg/L (nominal).
The ErC50 value for growth rate was determined to be 1.6 mg/L (nominal)
The overall NOEC was determined to be 0.320 mg/L (nominal), the corresponding LOEC was 0.8 mg/L (nominal).

Reported statistics and error estimates:

Potassium dichromate is tested as the toxic reference item in a separate study twice a year to confirm the sensitivity of the test organism against compounds with known effects under the test conditions. The EC50 values calculated in this reference test were considered to be within an acceptable range therefore it can be considered that the test organism is sensitive.

Toxicological endpoints were evaluated using nominal concentrations of the test item.
Due to an inhibition below 10% the database was weak for probit analysis which hence was not performed
No LOEC was observed

Any other information on results incl. tables

Validity Criteria of the Study:

Biomass	Cell numbers, measured in the controls between 0 h and 72 hours, were found to increase by a factor of 101.42, which exceeds the threshold of 16. It corresponds to a growth rate of 1.54104d^-1.
Coefficient of Variation (section by section)	The mean coefficient of variation for the section-by-section specific growth rates (hours 0 - 24, 24 - 48 and 48 - 72) in the control cultures was 25 % and did not exceed 35 %.
Coefficient of Variation (average growth rate)	The coefficient of variation of average growth rate in replicate control cultures was 2.7% and did not exceed 7 % for the whole test period.

Table 1: Average cell numbers for each sampling time and concentration

Conc.	Average cell numbers [10^4/mL]
[mg/L]	0 h	24 h	48 h	72 h
Control	0.57	1.78	11.65	57.81
0.954	0.57	2.05	12.37	61.48
3.05	0.57	2.09	13.26	63.52
9.77	0.57	2.12	15.40	64.70
31.3	0.57	1.90	16.31	80.79
100	0.57	1.81	13.24	81.81

Table 2: Percentage inhibition of growth rate

Conc.	% Inhibition of growth rate
[mg/L]	0 h – 24 h	0 h – 48 h	0 h – 72 h
Control	0.0	0.0	0.0
0.954	-11.6	-1.8	-1.1
3.05	-13.6	-4.2	-2.0
9.77	-14.7	-9.1	-2.2
31.3	-5.3	-11.1	-7.1
100	-0.9	-4.1	-7.4

Table 3: Percentage inhibition of yield

Conc.	% Inhibition of yield
[mg/L]	0 h – 24 h	0 h – 48 h	0 h – 72 h
Control	0.0	0.0	0.0
0.954	-22.3	-6.5	-6.4
3.05	-25.6	-14.5	-10.0
9.77	-28.1	-33.8	-12.0
31.3	-9.9	-42.1	-40.1
100	-2.5	-14.4	-41.9

 

Table 4: pH measurements during the test

Conc.	t = 0 h	t = 72 h
[mg/L]
Control	7.32	7.63
0.954	7.51	7.72
3.05	7.51	7.68
9.77	7.49	7.75
31.3	7.48	7.76
100	7.50	7.72

Table 5: Temperature during the test

Time	Temperature [°C]
[h]	min	max
0	22.5
24	23.0	23.3
48	22.5	23.3
72	22.5	23.3

Table 6: Individual cell numbers

Conc.	Cell numbers [10^4/mL]				Yield
[mg/L]	0 h	24 h	48 h	72 h	0 h – 72 h
	0.58	1.66	10.09	61.21	60.63
	0.67	1.75	11.91	56.73	56.06
Control	0.57	1.77	11.58	53.61	53.04
	0.59	1.81	11.97	57.90	57.31
	0.52	1.89	12.01	60.91	60.39
	0.49	1.82	12.32	56.49	56.00
Mean	0.57^1)	1.78	11.65	57.81	57.24
	0.57	2.12	12.25	63.80	63.23
0.954	0.57	1.81	11.34	53.44	52.87
	0.57	2.23	13.52	67.19	66.62
Mean	0.57	2.05	12.37	61.48	60.91
	0.57	2.13	13.55	63.48	62.91
3.05	0.57	2.02	13.52	62.93	62.36
	0.57	2.13	12.72	64.16	63.59
Mean	0.57	2.09	13.26	63.52	62.95
	0.57	2.12	14.45	54.45	53.88
9.77	0.57	2.10	15.58	71.31	70.74
	0.57	2.14	16.18	68.34	67.77
Mean	0.57	2.12	15.40	64.70	64.13
	0.57	1.82	16.49	82.90	82.33
31.3	0.57	1.95	15.91	74.13	73.56
	0.57	1.94	16.53	85.33	84.76
Mean	0.57	1.90	16.31	80.79	80.22
	0.57	1.81	14.41	86.65	86.08
100	0.57	1.85	12.28	75.94	75.37
	0.57	1.77	13.02	82.85	82.28
Mean	0.57	1.81	13.24	81.81	81.24

¹⁾The mean cell density of all control replicates is used as initial cell density for all treatment groups

Table 7: Control, growth rates

	Growth rates, µ [d^-1]
	0 h – 24 h	0 h – 48 h	0 h – 72 h
	1.05154	1.42814	1.55301
	0.96009	1.43893	1.47959
Control	1.13310	1.50570	1.51462
	1.12096	1.50502	1.52878
	1.29050	1.56983	1.58777
	1.31219	1.61229	1.58247
Mean	1.14473	1.50999	1.54104
Std. Dev.	0.13621	0.07189	0.04164
CV [%]	11.9	4.8	2.7

Std.Dev.:Standard deviation

CV: Coefficient of variation

Table 8: Control, daily growth rates

	Growth rates, µ [d^-1]
	0 h – 24 h	24 h – 48 h	48 h – 72 h
	1.05154	1.80473	1.80277
	0.96009	1.91776	1.56092
Control	1.13310	1.87830	1.53246
	1.12096	1.88908	1.57631
	1.29050	1.84916	1.62366
	1.31219	1.91239	1.52284
Mean	1.14473	1.87524	1.60316
Std. Dev.	0.13621	0.04252	0.10413
CV [%]	11.9	2.3	6.5

Std. Dev.:Standard deviation

CV: Coefficient of variation

Table 9: Coefficient of variation (CV) of controls’ daily growth rates

Mean daily growth rates µ [d^-1]		Std. Dev. [d^-1]	CV [%]
1.55301		0.43429	28
1.47959		0.48399	33
1.51462		0.37292	25
1.52878		0.38626	25
1.58777		0.28105	18
1.58247		0.30451	19
		Mean CV	25

Std. Dev.:Standard deviation

CV: Coefficient of variation

Table 10: Calibration data for cell numbers

Cell number [10^4/mL]^1)	Fluorescence measurement^2)				Mean	Std. Dev.	CV [%]
	Replicate 1	Replicate 2	Replicate 3	Replicate 4
104.50	14670.25	14725.25	14376.25	14296.25	14517.00	212.44	1.46
52.00	7541.25	7692.25	7688.25	7407.25	7582.25	136.19	1.80
26.00	4048.25	3905.25	3983.25	3826.25	3940.75	96.15	2.44
12.25	1950.25	2033.25	1853.25	1945.25	1945.50	73.56	3.78
7.13	1036.25	976.25	972.25	949.25	983.50	37.12	3.77
3.13	493.25	458.25	516.25	500.25	492.00	24.47	4.97
1.81	249.25	241.25	266.25	238.25	248.75	12.56	5.05
0.81	116.25	123.25	122.25	126.25	122.00	4.19	3.43
0.50	69.25	63.25	67.25	98.25	74.50	16.03	21.52
0.25	39.25	28.25	37.25	36.25	35.25	4.83	13.70
0.00	0.25	-0.75	0.25	0.25	0.00	0.50	-^3)

¹⁾The cell numbers were counted with a Neubauer chamber

²⁾Measured with a TECAN Reader

³⁾Not calculable due to a mean of zero

Std. Dev.:Standard deviation

CV: Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

No statistically significant inhibitory effects on any parameter (growth rate, yield) were observed at any of the test item concentrations, including the highest test item concentration of 100 mg/L (nominal) at test end. Thus the overall LOEC was not determinable and the overall NOEC was set at 100 mg/L (nominal).
Due to an inhibition of all parameters (growth rate, yield) below 10 % the database was weak for probit analysis, hence the EC10-, EC20- and the EC50-value for all parameters was considered to be > 100 mg/L (nominal).

Executive summary:

Study Objective:

The objective of this study was to determine the effects of the test item on the growth of the single cell green algae Pseudokirchneriella subcapitata, to determine the no observed effect concentration (NOEC), to determine the lowest observed effect concentration (LOEC) and to determine the effect concentration (EC10, 20, 50), where possible.

Material and methods:

Test item: Escherichia coli dehalogenase catalyst, batch number: 4898315, active ingredient (a.i.): (R)-2-haloacid halidohydrolase, dehalogenase activity: 120 U/g, total viable count 20 Cfu/g, coliform < 10 Cfu/g.

Test species: Pseudokirchneriella subcapitata.

Test design: Initial target cell densities of 0.5 x 10⁴ cells/mL were employed for the individual replicates. The increase of cell numbers was assessed over a test period of 72 hours.

Endpoints: Where possible inhibition of growth was assessed by the determination of NOEC/LOEC and EC10, 20, 50 for growth rate and yield after 72 hours.

Test rates: A static test with nominal test item concentrations of 100, 31.3, 9.77, 3.05, 0.954 mg/L and control was performed.

Test conditions: Six replicates were employed for the control and three for each test item concentration. An additional replicate per treatment without algae was incubated for the analytical sampling. The test was performed in 100 mL Erlenmeyer flasks each containing~ 50 mL test solution. The pH was recorded at test start and test end. Temperature was measured continuously over the whole test period and recorded daily. Light intensity of the continuous illumination was measured at test start.

Analysis: Analytical samples were taken from control and all test item concentrations at 0 hours (initial value) from fresh test solutions and after 72 hours from aged test solutions. The samples from the control, 9.77, 31.3 and 100 mg/L were analysed.

Statistics: NOEC and LOEC were determined by using a multiple comparison method.

Findings:                    

Validity criteria: Biomass: Cell numbers, measured in the controls between 0 h and 72 hours, were found to increase by a factor of 101.42, which exceeds the threshold of 16. It corresponds to a growth rate of 1.54104 d-1.

Coefficient of Variation (section by section): The mean coefficient of variation for the section-by-section specific growth rates (hours 0 - 24, 24 - 48 and 48 - 72) in the control cultures was 25 % and did not exceed 35 %.

Coefficient of Variation (average growth rate): The coefficient of variation of average growth rate in replicate control cultures was 2.7 % and did not exceed 7 % for the whole test period.

Test conditions: The pH-value of the control ranged from 7.32 to 7.63 during the test period, the temperature was measured to be 22.5 – 22.3 °C during the test and the mean light intensity was 88.1 µEm-2s-1at cell culture level.

Analytical Results: The measured initial TOC content ranged from 81 % to 108 % of nominal. In the aged samples the measured TOC content was between 80 % and 112 %. Therefore toxicological endpoints were evaluated using the nominal concentrations of the test item.

Statistical Results: Toxicological endpoints for the test item

	Test item [mg/L]
	nominal
ErC10(Growth rate)^1)	> 100
ErC20^1)	> 100
ErC50^1)	> 100
EyC10(Yield)^1)	> 100
EyC20^1)	> 100
EyC50^1)	> 100
NOEC^2)	100
LOEC^2)	-

¹⁾Due to an inhibition below 10% the database was weak for probit analysis which hence was not performed

²⁾Following Dunnetts-t-test (left-sided, p<0.05)for growth rate and for yield

- No LOEC was observed

Conclusions:

No statistically significant inhibitory effects on any parameter (growth rate, yield) were observed at any of the test item concentrations, including the highest test item concentration of 100 mg/L (nominal) at test end. Thus the overall LOEC was not determinable and the overall NOEC was set at 100 mg/L (nominal).
Due to an inhibition of all parameters (growth rate, yield) below 10 % the database was weak for probit analysis, hence the EC10-, EC20- and the EC50-value for all parameters was considered to be > 100 mg/L (nominal).