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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 25-JUN-2004 to 05-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 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

Oxygen concentrations fell below 5 mg/l at the end of the refreshment periods.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: from the limit concentration and the blank control
- Sampling method: 10 ml were taken from the approximate centre of the test vessels
- Sample storage conditions before analysis: Not applicable, samples were analysed on the day of sampling

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION: the test substance was dissolved in the test medium.
Since the test concentrations should be based on protein concentrations, the test solutions were prepared daily using stock solutions of 1230 mg/l. The stock solutions were magnetically stirred for 15 minutes to accelerate the dissolving of the test substance in the test medium. The lower test concentrations for the range-finding test were prepared by subsequent dilutions of the stock in test medium. The final test solutions were all clear and colourless with a thin layer of froth.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: carp
- Strain: Carp (Cyprinus carpio, Teleostei, Cyprinidae) Linnaeus, 1758
- Source: Zodiac, proefacc, "De Haar Vissen", L.U. Wageningen, the Netherlands
- Age at study initiation: data not available
- Length at study initiation: mean length was 2.2 +/- 1 cm in the range finding test and 2.3 +/- 0.2 cm in the limit test
- Weight at study initiation: mean weight was 0.26 +/- 0.05 g in the range finding test and 0.34 +/- 0.10 g in the limit test
- Method of breeding: F1 from a single parent-pair bred in UV-treated water.
- Feeding during test: No feeding from 48 hours prior to the test and during the total test period

ACCLIMATION
- Acclimation period: At least 12 days after delivery
- Acclimation conditions: ISO-medium
- Type and amount of food: Trouvit
- Feeding frequency: daily
- Health during acclimation: In the batch of fish used for the test, mortality during the seven days prior to the start of the test was less than 5%

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

none

Hardness:

After aeration the hardness was 250 mg CaC03 per litre

Test temperature:

20.6 to 21.5°C (20-24°C, constant within 2°C)

pH:

from 6.9 to 7.9 (6.0-8.5, constant within 1 unit)

Dissolved oxygen:

blank: from 7.3 to 9.0 mg/l
limit test concentration: from 0.5 to 9.0
See table 1

Salinity:

not applicable (freshwater)

Nominal and measured concentrations:

nominal: 0 and 1230 mg Aldolase/l
mean measured: 0 and 940 mg Aldolase/l
see table 2

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (open / closed): data not available
- Material, size, headspace, fill volume: 10.5 liters, all-glass, containing 9 liters of test medium
- Aeration: Aeration was introduced after 44.5 hours of exposure and was maintained for 28 hours. Aeration was introduced again after 93 hours of exposure and was maintained until the end of the test period.

- Renewal rate of test solution: renewal of test solutions after each 24-hour test period
- No. of organisms per vessel: 7 fish per concentration and control
- No. of vessels per concentration (replicates): one
- No. of vessels per control (replicates): one
- Biomass loading rate: 0.26 g fish/liter, i.e. 7 fish per 9 liter of test medium


TEST MEDIUM / WATER PARAMETERS: ISO-medium

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours photoperiod daily
- Light intensity: data not available


EFFECT PARAMETERS MEASURED:
- Mortality and other effects: at 3, 24, 48, 72 and 96 hours following the start of exposure
- Fish length and weight: ten fish of the batch used for the test, were weighed and measured prior to the start of the test
- Dissolved oxygen content, pH and temperature: Daily in all vessels, beginning at the start of the test (day 0)


TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable (limit test)
- 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: no mortality was observed after 96h of exposure

Reference substance (positive control):

yes

Remarks:

pentachlorophenol

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 940 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

Aldolase

Basis for effect:

mortality (fish)

Remarks on result:

other: no mortality observed

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 76 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: protein content

Basis for effect:

mortality (fish)

Remarks on result:

other: no mortality observed

Details on results:

No mortality was observed at the limit concentration or in the blank-control. However, at the last day of exposure all fish in the limit concentration were observed to be snapping at the surface.

Results with reference substance (positive control):

- Results with reference substance valid? yes
The 96h-LC50 for carp exposed to PCP was 0.32 mg/l (95 % confidence interval between 0.22 and 0.46 mg/l) and already reached within 24 hours of exposure. The range of the 96h- LC50 for carp is generally between 0.10 and 0.46 mg/l based on historical data of reference tests performed approximately every 3 months from April 1988 until the end 2000, and annually since then. The response observed in carp originating from the present batch falls within this range.

Reported statistics and error estimates:

not applicable

Sublethal observations / clinical signs:

Table 3: Incidence of mortality and total mortality during the limit test

Conc. Aldolase (mg/l)	Conc. Protein (mg/l)	Initial number of fish	Cumulative mortality					Total mortality (%)
			3h	24h	48h	72h	96h
Blank-control	Blank-control	7	0	0	0	0	0	0
1230	100	7	0	0	0	0	0	0

Validity criteria fulfilled:

no

Remarks:

Concentration at the end of the last refreshment period had decreased to 40% of nominal. Oxygen concentrations fell below 5 mg/l at the end of the refreshment periods. However the study was considered as valid.

Conclusions:

The 96h-LC50 was above an average exposure concentration of 940 mg Aldolase per liter, i.e above a protein concentration of 76 mg/l.

Executive summary:

In a 96-h acute toxicity study, carps (Cyprinus carpio) were exposed to Aldolase at nominal concentrations of 0 (control) and 1230 mg/l (i.e. 100 mg protein/l) under semi-static conditions.

The 96h-LC50 was above an average exposure concentration of 940 mg Aldolase per liter, i.e above a protein concentration of 76 mg/l. The 96h-NOEC was below and average concentration of 940 mg Aldolase per liter.

Aldolase induced no lethal effects in carp at an average exposure concentration of 940 mg/l, i.e. a protein concentration of 76 mg/l. However, at the end of the 96-hour test period fish were visibly affected (snapping at the surface) by the decrease of oxygen in the test solution due to the decay of the protein during refreshment periods.

The study generally met the acceptability criteria prescribed by the protocol and was considered valid. This can be stated in spite of the fact that oxygen concentrations fell below 5 mg/l at the end of the refreshment periods.

Reference 2

Endpoint:

short-term toxicity to fish

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:

96 h

Dose descriptor:

LC50

Effect conc.:

> 940 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

Aldolase

Basis for effect:

mortality (fish)

Remarks on result:

other: no mortality observed

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 76 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: protein content

Basis for effect:

mortality (fish)

Remarks on result:

other: no mortality observed

Conclusions:

The key value retained for the assessment of the toxicity towards fish of the target substance is a LC50 (96h) above an average exposure concentration of 940 mg Aldolase per liter, i.e above a protein concentration of 76 mg/L. (OECD 203, Cyprinus carpio, 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 fish 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 96 h acute toxicity study, carps (Cyprinus carpio) were exposed to Aldolase at nominal concentrations of 0 (control) and 1230 mg/L (i.e. 100 mg protein/L) under semi-static conditions.

The 96 h-LC50 was above an average exposure concentration of 940 mg Aldolase per liter, i.e above a protein concentration of 76 mg/L. The 96h-NOEC was below and average concentration of 940 mg Aldolase per liter.

Aldolase induced no lethal effects in carp at an average exposure concentration of 940 mg/L, i.e. a protein concentration of 76 mg/L. However, at the end of the 96-hour test period fish were visibly affected (snapping at the surface) by the decrease of oxygen in the test solution due to the decay of the protein during refreshment periods.

The study generally met the acceptability criteria prescribed by the protocol and was considered valid. This can be stated in spite of the fact that oxygen concentrations fell below 5 mg/L at the end of the refreshment periods.

ALDOLASE would not be classified as dangerous to fish (carp) under acute exposure conditions, according to EU criteria.

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

LC50 (96h) > 940 mg/L (i.e. > 76 mg protein content/L) (Cyprinus carpio, OECD 203, measured, read-across from CAS 9026-97-5)

Additional information

Experimental data on the toxicity of Glutaryl Acylase IUBMB 3.5.1.93 (CAS 56645-46-6) on the toxicity towards fish 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 96 h acute toxicity study, carps (Cyprinus carpio) were exposed to Aldolase at nominal concentrations of 0 (control) and 1230 mg/L (i.e. 100 mg protein/L) under semi-static conditions.

The 96 h-LC50 was above an average exposure concentration of 940 mg Aldolase per liter, i.e above a protein concentration of 76 mg/L. The 96h-NOEC was below and average concentration of 940 mg Aldolase per liter.

Aldolase induced no lethal effects in carp at an average exposure concentration of 940 mg/L, i.e. a protein concentration of 76 mg/L. However, at the end of the 96-hour test period fish were visibly affected (snapping at the surface) by the decrease of oxygen in the test solution due to the decay of the protein during refreshment periods.

The study generally met the acceptability criteria prescribed by the protocol and was considered valid. This can be stated in spite of the fact that oxygen concentrations fell below 5 mg/L at the end of the refreshment periods.

ALDOLASE would not be classified as dangerous to fish (carp) under acute exposure conditions, according to EU criteria.

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.