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Long-term toxicity to fish

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Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 2012 - February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
no
GLP compliance:
yes
Remarks:
GLP certificate included in report
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 0.32, 1, 3.2, 10 and 32 mg test material/L
- Sampling method: duplicate aliquots (ca. 1mL) were removed from one replicate tank at each concentration on Days -4, -1, 4, 11, 18, 25 and 32.
- Sample storage conditions before analysis: frozen at - 20°C
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Due to the nature of the test item, it was not possible to add it to the test system in the state it was supplied. The test item was therefore refined to remove any inert particulate material. Multiple small aliquots of Savinase, Batch PPA25057 were removed and centrifuged to allow the determination of solid content. The bulk test item was then homogenised by stirring on a magnetic stirrer, followed by centrifugation to separate the solid and liquid components of the test item. The supernatant was then removed and frozen in batches for use on study. Using the known density (1.034533 g/mL) and solid content (0.04587 g/mL), a conversion factor was calculated and applied to all dose calculations to allow the study to be dosed at relative nominal concentration.
Preparation of stock solutions:
Stock solutions of Savinase, Batch PPA 25027 in 0.2 µm filtered deionised water were prepared daily throughout the pre-equilibration and test periods. Solutions were prepared by serial dilution (1:10 v/v) of two primary stock solutions of Savinase, Batch PPA 25057 (nominally 510 mg/mL and 159.3 mg/mL). The primary stock solutions were prepared in a 5 mL volume by dissolving a weighed amount of Savinase, Batch PPA 25057 in 0.2 µm filtered deionised water (range: 2549.53–2555.58 mg to prepare the 510 mg/mL stock solution and 795.58–796.93 mg to prepare the 159.3 mg/mL stock solution). Stock solutions were added to syringes, and the loaded syringes added to the infusion pumps at ca. 24 h intervals. Stock solutions were maintained in an incubator set at 4°C.

- Controls: Medium
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnow
- Strain: Pimephales promelas
- Source: Charles River
POST-HATCH FEEDING
- Start date: newly hatched
- Type/source of feed: Artemia salina nauplii (brine shrimp, first instar larvae) less than 24 hours old
- Start date: 1 week after hatch
- Type/source of feed: Nauplii less than 48 hours old
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
32 d
Hardness:
68.8 - 88.0 mg CaCO3/L
Test temperature:
24.1-25.8°C
pH:
6.33-7.38
Dissolved oxygen:
74.5 - 96.2%
Nominal and measured concentrations:
Nominal: 0.32, 1, 3.2, 10 and 32 mg test material/L
Measured: 0.1, 0.2, 1.4, 4.2 and 14.2 mg test material/L corresponding to 0.1, 0.2, 1.4, 4.2 and 14.2 mg active enzyme protein (aep)/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Test tanks (external dimensions 22 cm length x 15 cm height x 12 cm width) constructed from glass plates sealed together with non-toxic aquarium silicone adhesive were used for the test. An overflow hole, covered with a stainless steel mesh screen to prevent any loss of fry, was positioned at a height to provide a capacity of ca. 3 L.
Egg incubation chambers used in the test were 8 cm long glass cylinders (5 cm internal diameter) with a fine stainless steel mesh attached to the bottom with non-toxic aquarium silicone adhesive. The vessels were suspended in the test tanks by stainless steel wire, which in turn, was attached to a bar which oscillated upwards and downwards at a rate of 2-4 rpm per minute (controlled by a cam shaft mounted on an electric motor). This motion ensured that the embryos were continuously exposed to a gentle flow of water. Each test tank contained one oscillating incubation chamber.

- Renewal rate of test solution (frequency/flow rate): ca. 5 tank replacements every 24 hours
- No. of fertilized eggs/embryos per vessel: 20 per egg chamber
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted fresh water was used as the test and culture medium. This was prepared at Charles River, with high grade salts and reverse osmosis grade water. The salts were dissolved into a volume of deionised water (500 L) and this solution was metered into a flow of deionised water, to produce reconstituted freshwater based on the formula recommended by OECD Guideline 203.
- Total organic carbon: 2.073 - 3.160 mg/L
- Particulate matter: 0.00
- Alkalinity: 15 mg CaCO3/L
- Conductivity: 202 µS/cm
- Intervals of water quality measurement: Alkalinity, hardness and conductivity once every 7 days and TOC and particulate matter at the beginning and at the end of the test.


OTHER TEST CONDITIONS
- Photoperiod: light/dark period 16:8hours
- Light intensity: 521 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Hatching success: Incubation chambers in each tank were observed daily and the number of dead, diseased or infertile embryos were recorded and discarded to reduce the risk of microbial contamination to surviving embryos. In the definitive test, Day 0 post-hatch was determined to be the day on which 95% of the embryos in the control had hatched (Study Day 4).
Fry: Daily observations of mortality, appearance, behaviour and any abnormal effects were recorded.






Reference substance (positive control):
no
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
0.042 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
active enzyme protein (aep)
Basis for effect:
mortality
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
0.126 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
aep
Basis for effect:
mortality
Duration:
32 d
Dose descriptor:
EC50
Effect conc.:
0.21 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
aep
Basis for effect:
mortality
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.426 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
aep
Basis for effect:
number hatched
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
>= 0.426 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
aep
Basis for effect:
number hatched
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
0.042 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
aep
Basis for effect:
other: time to 95% hatch
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
0.126 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
aep
Basis for effect:
other: time to 95% hatch
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
0.042 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
aep
Basis for effect:
length
Remarks:
fry
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
0.126 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
aep
Basis for effect:
length
Remarks:
fry
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
0.042 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
aep
Basis for effect:
weight
Remarks:
fry
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
0.126 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
aep
Basis for effect:
weight
Remarks:
fry
Key result
Duration:
32 d
Dose descriptor:
EC10
Effect conc.:
0.017 mg/L
Nominal / measured:
estimated
Conc. based on:
act. ingr.
Remarks:
aep
Basis for effect:
mortality
Duration:
32 d
Dose descriptor:
EC10
Effect conc.:
0.6 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Mortality/survival at embryo, larval, juvenile, and adult stages: A significant effect on the larval mortality was observed at concentrations ≥0.64 mg/L (nominal ≥5 mg/L)
- Days to hatch or time to release of young: no effects
- Number of fish in swim-up stage at one or more time periods (e.g., day x1, x2): no effects
- Observations on body length and weight of young and/or exposed parents at one or more time periods: a significant inhibition of the growth rate was observed at 0.16 mg/L (nominal 2 mg/L)
- Number of healthy fish at end of test: control: 48 ; control: 52; 0.5 mg/L: 43; 1.0 mg/L: 49; 2.0 mg/L: 42; 5.0 mg/L: 33 ; 10 mg/L: 24
Validity criteria fulfilled:
yes
Conclusions:
The EC5, EC10, EC20 and EC50 values of Savinase, Batch PPA25057, measured as active enzyme protein (AEP) on mortality were estimated to be 0.009, 0.017, 0.041 and 0.210 mg AEP/L, respectively (equivalent to 0.3, 0.6, 1.4, and 7.0 mg Savinase, Batch PPA 25057/L, respectively).

It is concluded that the NOEC of Savinase, Batch PPA 25057, measured as AEP, on hatching success in fathead minnows is ≥0.426 mg AEP/L (equivalent to ≥14.2 mg Savinase, Batch PPA 25057/L). The NOEC for mortality, body length and weight and time to 95 % hatch is 0.042 mg AEP/L (equivalent to 1.4 mg Savinase, Batch PPA 25057/L)

Mortality, body length and weight and time to 95% hatch appear to be the most sensitive parameters in this early life stage test of the fathead minnow, with the NOEC for Savinase, Batch PPA 25057 being 0.042 mg AEP/L (equivalent to 1.4 mg Savinase, Batch PPA 25057/L).
Executive summary:

The effects of prolonged exposure to Savinase, Batch PPA 25057 on the early life stages of the fathead minnow (Pimephales promelas) were assessed over embryo development, hatching and for 28 days post-hatch, in accordance with OECD Guideline 210 (2013).

 

Based on the results of a range finding test, the definitive test was conducted under continuous flow conditions, with embryos (< 24 h old) exposed to the following nominal concentrations: 0.32, 1, 3.2, 10 and 32 mg/L of Savinase, Batch PPA 25057. A control was also included. Four replicate tanks were tested at each concentration.

 

Analysis of test samples taken during the exposure period indicated that measured concentration of Savinase, Batch PPA 25057 were not maintained within ±20% of nominal over the test period. Due to the nature of the test item and the analytical method results are reported in mg of active enzyme protein per litre of test solution (mg AEP/L). The geometric mean measured concentrations of AEP were determined to be 0.426, 0.126, 0.042, 0.006 and 0.002 mg AEP/L (equivalent to 14.2, 4.2, 1.4, 0.2 and 0.1 mg Savinase, Batch PPA 25057/L) for the respective nominal concentrations of 32, 10, 3.2, 1 and 0.32 mg/L.

 

The hatching success of embryos in individual replicates ranged from 75-100%. Hatching success in the control group was 92.5%. Statistical analysis indicated no significant differences in hatching success between the treatment groups and the control group. As there was no difference in hatching success between the treatment groups and the control; the no observed effect concentration (NOEC) for hatching success is concluded to be ≥0.426 mg AEP/L. The corresponding lowest observed effect concentration (LOEC) is concluded to be >0.426 mg AEP/L (equivalent to ≥14.2 and >14.2 mg Savinase, Batch PPA 25057/L, respectively).

 

Hatching commenced 2 days after the initiation of the test in all treatment groups. Time to 95% hatch in the control, 0.002, 0.006 and 0.042 mg AEP/L treatment groups was Day 4. In the 0.126 and 0.426 mg AEP/L treatment groups, time to 95% hatch was Day 3. The NOEC for time to hatch is concluded to be 0.042 mg AEP/L; the corresponding LOEC is concluded to be 0.126 mg AEP/L (equivalent to 1.4 and 4.2 mg Savinase, Batch PPA 25057/L).

 

Survival of fry at the end of the exposure phase was compared to the number of fry successfully hatched and released in individual replicates, and ranged from 0-100%. Survival of fry in the control group was 100%. Statistical analysis indicated a significantly higher mortality in the 0.126 and 0.426 mg AEP/L treatment groups than in the control. There was no significant difference in mortality between the 0.002, 0.006 and 0.042 mg AEP/L treatment groups and the control. The EC5, EC10, EC20and EC50values were estimated to be 0.009, 0.017, 0.041 and 0.210 mg AEP/L, respectively (equivalent to 0.3, 0.6, 1.4 and 7.0 mg Savinase, Batch PPA 25057/L, respectively). 

The NOEC for mortality is concluded to be 0.042 mg AEP/L; the corresponding LOEC is concluded to be 0.126 mg AEP/L (equivalent to 1.4 and 4.2 mg Savinase, Batch PPA 25057/L, respectively).

 

Fry length and body weight were measured at the end of the test. Statistical analysis indicated no significant differences in length and body weight between the treatment groups and the control. The NOEC is concluded to be 0.042 mg AEP/L for both body lengths and body weights; the corresponding LOEC is concluded to be 0.126 mg AEP/L (equivalent to 1.4 and 4.2 mg Savinase, Batch PPA 25057/L, respectively).

 

Based on the above results, mortality, body length and weight, and time to 95% hatch appear to be the most sensitive parameters in the early life stage test of the fathead minnow, with the NOEC for Savinase, Batch PPA 25057 being 0.042 mg AEP/L (equivalent to 1.4 mg Savinase, Batch PPA 25057/L).

 

Test solution quality parameters measured throughout the test were in the following ranges; temperature: 24.7-27.1°C, pH: 6.33-7.38 and dissolved oxygen: 74.5-96.2% ASV.

 

Validity criteria for the test was met, as the hatching success in the controls was greater than 70% (92.5%); survival of fry in the control group was greater than 75% (100%); dissolved oxygen concentration in each of the test tanks was above 60% ASV for the duration of the test (≥74.5% ASV) and the water temperature did not differ by more than ± 1.5°C between test tanks. As such, this test is regarded as valid.

Chronic aquatic classification is based on the EC10value of 17 µg aep/L as the EC10value in general is preferred compared to NOEC values for deriving long-term toxicity to aquatic organisms. In addition, the derived EC10value is below the NOEC value and thus the approach taken must be considered conservative.

Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
28 September 2007 - 5 November 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
no
GLP compliance:
yes
Remarks:
except for analytical analysis of the test material
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 0.5, 1.0, 2.0, 5.0 and 10 mg test product/L
- Sampling method: duplicate subsamples were taken at the start of the analysis and hereafter as a minimum once every week during the 33-day test period. Subsamples were collected in 12 mL Nunc-Immuno Minisorp Tubes
- Sample storage conditions before analysis: frozen at - 20°C
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions of the test by weighing out respectively 40, 80, 160, 400 and 800 mg of the test material and adding it to a 2 litre of the test medium. Stock solutions were prepared every second day througout the test period and placed in box with ice to keep the temperature below 10°C.
- Controls: Medium
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebra fish
- Strain: Danio rerio
- Source: Credo fish, Nørresundby, Denmark and purchased from Hørsholm Dyrehandel, Hørsholm, Denmark

POST-HATCH FEEDING
- Start date: newly hatched
- Type/source of feed: TetraMin®Baby (Tetra GmbH, Melle)
- Start date: 2 days-old
- Type/source of feed: TetraMin®Baby (Tetra GmbH, Melle) supplemented with newly hatched Artemia salina nauplii
- Start date: 2 weeks after hatch
- Type/source of feed: only Artemia salina nauplii
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
33 d
Test temperature:
24.4 - 25.7°C
pH:
6.9-7.4
Dissolved oxygen:
79-100%
Nominal and measured concentrations:
Nominal: 0.5, 1.0, 2.0, 5.0, 10 mg test material/L
Measured: 0.014, 0.042, 0.16, 0.64 and 1.24 mg test material/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 6-L glass aquaria
- Material, size, headspace, fill volume: glass, size: 6L, 3L fill volume
- Renewal rate of test solution (frequency/flow rate): 4 times daily
- No. of fertilized eggs/embryos per vessel: 30
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Freshly produced synthetic medium was used in the test. The medium was prepared from Millipore water according to the ISO Standard 7346/3/.


OTHER TEST CONDITIONS
- Adjustment of pH: adjusted with 1M HCl to be below 7.
- Photoperiod: light/dark period 14:10 hours

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : embryonic develpoment, hatching, survival, abnormal appearance and behaviour were observed at least once a day.

VEHICLE CONTROL PERFORMED: no


Reference substance (positive control):
no
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
0.16 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
larval
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
larval
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
6 µg/L
Nominal / measured:
estimated
Conc. based on:
act. ingr.
Remarks:
active enzyme protein
Basis for effect:
mortality
Remarks:
larval
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 1.24 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
embryonic
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 1.24 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
other: time to 50% hatching
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 1.24 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
other: Larval malformation and behaviour
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 1.24 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
other: larval growth
Details on results:
- Mortality/survival at embryo, larval, juvenile, and adult stages: A significant effect on the larval mortality was observed at concentrations ≥0.64 mg/L (nominal ≥5 mg/L)
- Days to hatch or time to release of young: no effects
- Number of fish in swim-up stage at one or more time periods (e.g., day x1, x2): no effects
- Observations on body length and weight of young and/or exposed parents at one or more time periods: a significant inhibition of the growth rate was observed at 0.16 mg/L (nominal 2 mg/L)
- Number of healthy fish at end of test: control: 48 ; control: 52; 0.5 mg/L: 43; 1.0 mg/L: 49; 2.0 mg/L: 42; 5.0 mg/L: 33 ; 10 mg/L: 24
Validity criteria fulfilled:
yes
Remarks:
The validation criteria of the test according to the OECD Test Guideline are fulfilled
Conclusions:
The no-observed-effect concentration (NOEC) for larval mortality of the test substance was nominally 2 mg/L. Based on the measured levels the NOEC for larval mortality was estimated to be 0.16 mg/L corresponding to 6.0 µg active enzyme protein/L.
Executive summary:

Subtilisin was tested for lethal and sublethal effects on early life stages of Zebra fish (Danio rerio) according to the procedures described in the OECD Test Guideline No. 210 and conducted in compliance with GLP.

The test material was tested at following nominal concentrations: 0, 0.5, 1.0, 2.0, 5.0 and 10 mg/L.

The test was carried out in a flow-through test system with 6 liter glass aquaria each containing 3 liter of test solutions. Effect parameters observed during the 33 -day test period included time to hatch, mortality, behavior and growth of early life stages of zebra fish.

The actual test concentrations were analysed by specific ELISA analysis. The analysis showed recoverys from 0 to 32 % indicating fast degradation of the test compound. Samples were kept frozen until analysis and the freeze thawing proces can also have contributed to the loss of enzymatic activity.

No effects were obtained for the following effect parameters: Time to 50% hatch, embryonic mortality, malformation, growth (length and weight) and swimming behavior.

Significant effect on the larval mortality was observed at estimated concentrations ≥0.64 mg/L ( nominal concentration: ≥5mg/L). The no-observed-effect-concentration (NOEC) for larval mortality was thus 0.16 mg/L (estimated concentration) corresponding to 6.0 µg aep/L and 2 mg/L nominal.

Due to the low recovery of the exposure concentration, the study must be regarded as supporting study.

Description of key information

The EC10 value from the early life stage study of fish was determined to be 0.017 mg active enzyme protein/L (geometric mean measured concentration).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
17 µg/L

Additional information

In total two early life stage tests have been performed, one with zebra fish (supporting study) and one with fathead minnow (key study). Both tests were performed according to OECD guidelines and in compliance with GLP.

In order to keep the exposure concentrations constant, the tests were conducted in flow through systems.

Due to problems with test material recovery in the supporting test (standard flow through design), the test design was optimized and adjusted to the speciel properties of subtilisin in the fathead minnow study. In the later not only the flow through system was designed and optimised in order to keep the exposure concentration constant througout the whole study period but also exposure analysis was performed on site immediatly after sampling in order to eliminate any inactivation/loose of test material during storage and transport. Thus it was possible at the end of the study to calculate effect concentrations, whereas effect concentrations only could be estimated in the study with zebra fish due to the very poor recovery (0 -32%). Hence, the study with fathead minnow was selected a skey studies and used for derivation of PNEC values.