(7R)-7-(4-carboxybutanamido)cephalosporanate amidohydrolase (enzyme substance)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 15-JUL-2004 to 09-AUG-2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The test was performed according to OECD guideline and GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Range-finding test:
> concentrations: 123 mg Aldolase/l
> Frequency: at t=0 h and t=72 h
> Volume: 10 ml
> Storage: Not applicable, samples were analysed on the day of sampling

Final tests:
> concentrations: samples were taken from all concentrations and the blank-control
> Frequency: at t=0 h and t=72 h (first EC50-test), at t=0 h, t=24 h and t=72 h (final EC50-test)
> Volume: 10 ml
> Storage: Not applicable, samples were analysed on the day of sampling

Vehicle:

no

Details on test solutions:

Since the test concentrations should be based on protein concentrations, the test solutions were prepared using a stock solution of 1230 mg/l to obtain a concentration of 100 mg protein per liter. The stock solution was magnetically stirred for 15 minutes to accelerate the dissolving of the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the stock in test medium. The final test solutions were all clear and colourless.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Selenastrum capricornutum
- Strain: NlVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum (at test initiation): 3 or 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 2.10E4 cells/ml. The pre-culture was maintained under the same conditions as used in the test.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 +/- 2°C.


ACCLIMATION
- Acclimation period: 3 or 4 days
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: data not available

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Hardness:

data not available

Test temperature:

- first EC50 test: between 23.6 and 23.9°C
- final EC50 test: between 23.7 and 24.3°C

pH:

- first EC50 test: The pH was generally within the limits prescribed by the protocol (6.0-9.0, preferably not varying by more than 1.5 unit). The pH of the blank-control and the lowest test concentration exceeded the upper limit at the end of the test due to a relatively high algal growth rate.
- final EC50 test: The pH of the blank control and all test concentrations exceeded the upper limit at the end of the test and some of the pH values also varied by more than 1.5 unit during the test period. This was due to a relatively high algal growth rate.

Dissolved oxygen:

data not available

Salinity:

not applicable (freshwater)

Nominal and measured concentrations:

First EC50 test:
- Nominal Aldolase concentration: 123, 220, 395, 690, 1230 mg/l
- Nominal protein concentration: 10, 18, 32, 56, 100 mg/l
- Average exposure concentration (mg Aldolase/l): 14, 43, 66, 124, 259 mg/l
- Average exposure concentration (mg protein/l): 1.2, 3.5, 5.3, 10, 21 mg/l

Final EC50 test:
- Nominal Aldolase concentration: 123, 220, 395, 690, 1230 mg/l
- Nominal protein concentration: 10, 18, 32, 56, 100 mg/l
- Average exposure concentration (mg Aldolase/l): 15, 83, 250394, 1042 mg/l
- Average exposure concentration (mg protein/l): 1.2, 6.7, 20, 32, 84 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (open / closed): data not available
- Material, size, headspace, fill volume: 100 ml, all-glass, containing 50 ml of test medium
- Aeration: no
- Initial cells density: 1 x 10E4 cells/ml
- Control end cells density: 67.1 x 10E4 cells/ml
- No. of vessels per concentration: 3 replicates of each test concentration
- No. of vessels per control: 6 replicates of the blank-control


GROWTH MEDIUM
- Standard medium used: yes (M2 medium)


OTHER TEST CONDITIONS
- Sterile test conditions: data not available
- Adjustment of pH: no
- Photoperiod: Continuously illuminated
- Light intensity and quality: light intensity within the range of 71 to 102 uE.m-2.s-1 (first EC50-test) or within the range of 79 to 106 uE.m-2.s-1 (final EC50-test)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: at 24, 48 and 72h, cells were counted using a microscope and a counting chamber


TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 1.8
- Range finding study
- Test concentrations: 1.23, 12.3, 123 and 1230 mg test substance per liter, containing 0.1, 1 .0, 10 and 100 mg protein per liter
- Results used to determine the conditions for the definitive study: the expected EC50 for cell growth inhibition was between nominal 123 mg and nominal 1230 mg Aldolase per liter

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 042 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

Aldolase

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 84 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: protein content

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 042 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

Aldolase

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: data not available
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: from 24 hours of exposure onwards test substance particles were observed at all test concentrations. An exception was the lowest test concentration in which test substance particles were observed from 48 hours of exposure onwards.

Results with reference substance (positive control):

The EC50 for growth rate reduction (ErC50: 0-72h) was 1.0 mg/l with a 95% confidence interval ranging from 0.58 to 1.8 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ErC50: 0-72h for the present batch corresponds with this range.

Reported statistics and error estimates:

not applicable

Table 1: Mean cell densitied (x 10E4 cells/ml) during the first EC50-test

Average conc. Aldolase (mg/l)	Average conc. Protein (mg/l)	Exposure time (hours)
		0	24	48	72
Blank control	Blank control	1.0	5.4	32.4	71.1
14	1.2	1.0	5.4	31.9	63.5
43	3.5	1.0	5.3	33.7	76.5
66	5.3	1.0	5.3	32.1	77.7
124	10	1.0	3.9	20.3	70.0
259	21	1.0	3.7	15.8	73.4

 

Table 2: Percentage inhibition of cell growth during the first EC50-test

Average conc. Aldolase (mg/l)	Average conc. Protein (mg/l)	Cell growth (0-72 hrs)
		Mean area (A)	Inhibition (%)
Blank control	Blank control	1700.50
14	1.2	1598.00	6.0
43	3.5	1794.00	-5.5
66	5.3	1770.00	-4.1
124	10	1362.00	19.9
259	21	1289.00	24.2

 

Table 3: Mean cell densitied (x 10E4 cells/ml) during the final EC50-test

Average conc. Aldolase (mg/l)	Average conc. Protein (mg/l)	Exposure time (hours)
		0	24	48	72
Blank control	Blank control	1.0	6.5	40.7	67.1
15	1.2	1.0	4.9	38.8	67.8
83	6.7	1.0	6.9	37.3	70.0
250	20	1.0	6.4	40.6	69.0
394	32	1.0	6.4	37.9	74.7
1042	84	1.0	3.0	39.9	69.7

 

Table4: Percentage inhibition of cell growth during the final EC50-test

Average conc. Aldolase (mg/l)	Average conc. Protein (mg/l)	Cell growth (0-72 hrs)
		Mean area (A)	Inhibition (%)
Blank control	Blank control	1878.00
15	1.2	1801.00	4.1
83	6.7	1842.00	1.9
250	20	1896.00	-1.0
394	32	1900.00	-1.2
1042	84	1806.00	3.8

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Selenastrum capricornutum, no inhibition of cell growth or reduction of growth rate was recorded at any of the concentrations of Aldolase tested. ErC50 > 1042 mg Aldolase/l (i.e. 84 mg protein/l).

Executive summary:

In a 72 hour acute toxicity study, the cultures of Selenastrum capricornutum were exposed to Aldolase at 1.2, 6.7, 20, 32 and 84 mg protein per liter, corresponding to 15, 83, 250, 394 and 1042 mg Aldolase per liter, under static conditions in accordance with the OECD guideline 201 and GLP.

The EC50 for both cell growth inhibition (EbC50: 0-72h) and growth rate reduction (ErC50: 0-72h) was beyond an average exposure concentration of 84 mg protein per liter, i.e. beyond an average exposure concentration of 1042 mg Aldolase per liter. NOEC was 1042 mg ALdolase per liter.

This toxicity study is classified as acceptable and satisfies the guideline requirements for algal toxicity study.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to the analogue justification attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 042 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

Aldolase

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 84 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: protein content

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 042 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

Aldolase

Basis for effect:

growth rate

Conclusions:

Under the conditions of the present study with Selenastrum capricornutum, no inhibition of cell growth or reduction of growth rate was recorded at any of the concentrations of Aldolase tested. The key values retained for the assessment of the toxicity towards aquatic algae and cyanobacteria of the target substance are an ErC50 (72h) > 1042 mg Aldolase per liter (i.e 84 mg protein/L) and a NOEC of 1042 mg Aldolase per liter (i.e 84 mg protein/L). (OECD 201,Selenastrum capricornutum, RA to CAS 9026-97-5).

Executive summary:

Experimental data on the toxicity of Glutaryl Acylase IUBMB 3.5.1.93 (CAS 56645-46-6) on the toxicity towards aquatic algae and cyanobacteria are not available. The toxicity was assessed using read across data from the analogue substance Aldolase enzyme concentrate; deoxyribose-phosphate aldolase IUB 4.1.2.4 (CAS 9026-97-5). This read-across is in accordance with Regulation (EC) No. 1907/2006 Annex XI, 1.5. Details on the read across justification are provided in section 13 of the IUCLID dossier.

In a 72 hour acute toxicity study, the cultures of Selenastrum capricornutum were exposed to Aldolase at 1.2, 6.7, 20, 32 and 84 mg protein per liter, corresponding to 15, 83, 250, 394 and 1042 mg Aldolase per liter, under static conditions in accordance with the OECD guideline 201 and GLP.

The EC50 for both cell growth inhibition (EbC50: 0-72h) and growth rate reduction (ErC50: 0-72h) was beyond an average exposure concentration of 84 mg protein per liter, i.e. beyond an average exposure concentration of 1042 mg Aldolase per liter. NOErC was 1042 mg ALdolase per liter.

This toxicity study is classified as acceptable and satisfies the guideline requirements for algal toxicity study.

In conclusion, this result is considered for Glutaryl Acylase IUBMB 3.5.1.93 (CAS 56645-46-6), too, based on the read-across conducted.

Description of key information

EbC50 (72h) and ErC50 (72h) > 1042 mg/L (i.e. 84 mg protein content/L) (Pseudokirchneriella subcapitata, OECD 201, measured, read-across from CAS 9026-97-5)

NOErC (72h) = 1042 mg/L (i.e. 84 mg protein content/L) (Pseudokirchneriella subcapitata, OECD 201, measured, read-across from CAS 9026-97-5)

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

1 042 mg/L

Additional information

Experimental data on the toxicity of Glutaryl Acylase IUBMB 3.5.1.93 (CAS 56645-46-6) on the toxicity towards aquatic algae and cyanobacteria are not available. The toxicity was assessed using read across data from the analogue substance Aldolase enzyme concentrate; deoxyribose-phosphate aldolase IUB 4.1.2.4 (CAS 9026-97-5). This read-across is in accordance with Regulation (EC) No. 1907/2006 Annex XI, 1.5. Details on the read across justification are provided in section 13 of the IUCLID dossier.

In a 72 hour acute toxicity study, the cultures of Selenastrum capricornutum were exposed to Aldolase at 1.2, 6.7, 20, 32 and 84 mg protein per liter, corresponding to 15, 83, 250, 394 and 1042 mg Aldolase per liter, under static conditions in accordance with the OECD guideline 201 and GLP.

The EC50 for both cell growth inhibition (EbC50: 0-72h) and growth rate reduction (ErC50: 0-72h) was beyond an average exposure concentration of 84 mg protein per liter, i.e. beyond an average exposure concentration of 1042 mg Aldolase per liter. NOEC was 1042 mg ALdolase per liter.

This toxicity study is classified as acceptable and satisfies the guideline requirements for algal toxicity study.

In conclusion, this result is considered for Glutaryl Acylase IUBMB 3.5.1.93 (CAS 56645-46-6), too, based on the read-across conducted.