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Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
14 November 2007 - 5 December 2007
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD guideline 211, but subtilisin could not be detectet in the test media after 48 hours and thus the actual exposure concentration could not be determined.
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
The test was performed without bicine control. The deviation is not considered to be significant nor to have affected the integrity of the test.
GLP compliance:
yes
Remarks:
except for the analysis of the test material
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 0.03, 0.1, 0.3, 1.0, 3.0 and 10 mg/L
- Sampling method: Duplicate samples of 2 x 10 mL in Nunc-Immuno Minisorp Tubes were collected from the stock solutions and from the test solutions 1.0, 3.0 and 10 mg/L. The subsamples were frozen to -20°C immediately after collection.
- Sample storage conditions before analysis: Frozen at appr. -20°C
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions of 100 mg/L of the test substance were prepared by weighing 25 mg and adding it to 250 mL of test medium. This procedure was followed at the beginning of the test and at each renewal of test medium, i.e. every second day during the test period. The stock solutions were used for preparing a dilution series of the test product in the test medium.
- Controls: medium.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus
- Source: Langedam, Birkerød
- Feeding during test
- Food type: alga Pseudokirchneriella subcapitata
- Amount: appr. 0.2 mgC/animal/day until day 8, herafter 0.4 mgC/animal/day
- Frequency: daily
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Test temperature:
19.5 ± 0.1°C
pH:
6.1-7.9
Dissolved oxygen:
98-100%
Nominal and measured concentrations:
nominal: 1.0, 3.0 and 10 mg/L
Measured: Subtilisin could not be detected in expired media. In freshly prepared media the measured concentrations ranged between 4 and 76% of the expected concentration.

Details on test conditions:
TEST SYSTEM
- Test vessel: glass flask
- Material, size, headspace, fill volume: glass, 120-mL, fill volume 80 mL
- Renewal rate of test solution (frequency/flow rate): every second day
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10


OTHER TEST CONDITIONS
- Adjustment of pH: to pH 6.2 ± 0.2 using either HCl or NaOH
- Photoperiod: 16 hours light: 8 hours dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Number of offspring, mortality among parent animals, time at which the first offspring were present, aborted broods and dead larvae

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: 1, 3 and 10 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.021 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.8 µg/L
Nominal / measured:
estimated
Conc. based on:
act. ingr.
Remarks:
aep
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
> 0.021 - < 0.071 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
> 0.3 - < 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 0.3 - < 1.5 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 3 - < 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
- Mortality of parent animals: control: 1, 0.03mg/L: 1, 0.1 mg/L: 1, 0.3 mg/L: 3; 1.0 mg/L: 1; 3.0 mg/L: 2; 10 mg/L: 9
- No. of offspring produced per day per female: control: average 2.9-31.7; 0.03 mg/L: average 2.6-29.7; 0.1 mg/L: average 0.8 - 22; 0.3 mg/L: average 0-30.2; 1.0 mg/L: average 1-10.9; 3 mg/L: average 0-23.8; 10 mg/L: 0-14
- Number of males and females (parental): 10 females per group
- Time to first brood release or time to hatch: 9 days in both control and all test concentrations

Validity criteria fulfilled:
yes
Conclusions:
The test material could not be detected in the test media after 48 hours of exposure and thus it was not possible to determine the exposure concentration in the test media. Based on nominal concentrations the NOEC was determined to be 0.3 mg/L.
Executive summary:

A Daphnia magna reproduction test has been performed according to OECD guideline 211, and in compliance with GLP. The test performed was a semi-static test with medium renewal every second day. Freshly prepared and 48 hour-old test media were analysed for test material. No test material could be detected in 48 hour-old test media indicating fast degradation of the test substance in the media. Thus it was not possible to make an accurate estimation of the exposure concentrations. However, estimates of exposure concentration have been made based on time-weighted mean.

The data processing showed that NOEC-21d was 0.021 mg/L (nominal 0.3 mg/L), EC10 -21d was estimated to be between 0.021 mg/L (nominal 0.3 mg/L) and 0.071 mg/L (nominal 1.0 mg/L). The EC50 -21d was estimated to be between 0.3 mg/L (nominal 3.0 mg/L) and 1.5 mg/L (10 mg/L).

Endpoint:
long-term toxicity to aquatic invertebrates
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 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 0.32, 1, 3.2, 10 and 32 mg test material/L
- Sampling method: From each treatment vessel samples have been taken on at least two occasions prior to the addition of Daphnia magna to the test system and on 4 at least 4 occasions during the study.
- Sample storage conditions before analysis: Samples have been analysed directly after collection in order to avoid degradation of the test material.
Vehicle:
no
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus
- Source: From a culture maintained within Charles River bred by acyclical parthogenesis and as such will be female.
- Feeding during test
- Food type: microalga Chlorella vulgaris
- Amount: 0.1 - 0.2 mg C/Daphnia/day

Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Test temperature:
18-22°C
Dissolved oxygen:
> 3 mg/L
Nominal and measured concentrations:
Nominal: 32, 10, 3.2, 1 and 0.32 mg test material/L
Measured: 0.003, 0.006, 0.019, 0.163 and 0.324 mg active enzyme protein (aep)/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass
- Type (delete if not applicable): open
- Material, size, fill volume: glass, ca. 2 L, ca. 2 L
- Type of flow-through : A single glass tank (22 cm x 15 cm X 12 cm, length x width X height) for each treatment with an overflow hole providing ca. 2 litre of liquid capacity. Suspended within this tank were 4 glass chambers, constructed from 8 cm long glass cylinders (5 cm internal diameter) with a fine mesh steenless steel screen attached to the base.
- Renewal rate of test solution (flow rate): 5- 10 volume changes in a 24h period
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium
- Total organic carbon: <=2 mg/L

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark
- Light intensity: < 1600 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

RANGE-FINDING STUDY
- Test concentrations: 0.01, 0.1, 1 and 10 mg test material/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
no
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
0.019 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
aep
Basis for effect:
reproduction
Duration:
14 d
Dose descriptor:
EC10
Effect conc.:
0.052 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
aep
Basis for effect:
reproduction
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
0.221 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
aep
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.324 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
aep
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
0.145 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
aep
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
other: EC20
Effect conc.:
0.292 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
aep
Basis for effect:
reproduction
Validity criteria fulfilled:
yes
Conclusions:
The Day 14 EC5, EC10, EC20 and EC50 (reproduction) of Savinase, Batch PPA 25057 in D. magna, was concluded to be 0.035, 0.052, 0.086 and 0.221 mg AEP/L (equivalent to 1.2, 1.7, 2.9 and 7.4 mg Savinase, Batch PPA 25057/L), respectively, based on geometric mean measured concentrations, under the conditions of the test.

The Day 21 EC5,EC10 and EC20 (reproduction) of Savinase, Batch PPA 25057 in D. magna, were concluded to be 0.082, 0.145 and 0.292 mg AEP/L (equivalent to 2.7, 4.8 and 9.7 mg Savinase, Batch PPA 25057/L), based on geometric mean measured concentrations, under the conditions of the test.

The Day 14 and 21 NOEC (reproduction) of Savinase, Batch PPA 25057 were determined as 0.019 and 0.324 mg AEP/L (equivalent to 0.6 and 10.8 mg Savinase, Batch PPA 25057/L) based on geometric mean measured concentrations, under the conditions of the test.
Executive summary:

This study was designed to determine the effect of Savinase, Batch PPA 25057 on the reproductive capacity of Daphnia magna. The study was conducted over a 21 day period under flow through conditions. Four replicate vessels, contained within a tank of test solution, were prepared for each treatment group, each containing 5D. magna neonates. The study was performed to meet the requirements of OECD Guideline 211 (2012).

Based on the results of a range finding test, the reproduction test was conducted under flow through conditions in Elendt M4 medium, with neonates being exposed to the following nominal concentrations of Savinase, Batch PPA 25057: 0.32, 1, 3.2, 10 and 32 mg/L. A control of untreated Elendt M4 medium was also included. As the test item is an unstable biological preparation, the method of exposure was selected to maximise the stability of Savinase, Batch PPA 25057 under test conditions.

 

Analysis of test samples taken during the exposure period indicated that measured concentrations of Savinase, Batch PPA 25057, were not maintained within ±20% of nominal over the test period due to the unstability of the test substance in aquatic media and thus results are based on measured and not nominal concentrations. Due to the nature of the test item and the analytical method results are reported as mg active enzyme protein (AEP)/L. The geometric mean measured concentrations of AEP in Savinase, Batch PPA 25057, were calculated as 0.324, 0.163, 0.019, 0.006 and 0.003 mg/L (equivalent to 10.8, 5.4, 0.6, 0.2 and 0.1 mg Savinase, Batch PPA 25057/L) for the nominal concentrations of 32, 10, 3.2, 1 and 0.32 mg of Savinase, Batch PPA 25057/L respectively.

The number of immobile adult D. magnarecorded during the reproduction test was 1 (5%) in the control and 0.003 mg AEP/L treatment groups. In the 0.019 and 0.163 mg AEP/L treatment groups, there were 3 (15%) and 5 (25%) immobile adult D. magna recorded, respectively. There were no immobile adultD. magna observed in the 0.006 and 0.324 mg AEP/L treatment groups.

 

The total number of offspring produced by surviving D. magna at each concentration and the mean number of offspring produced per surviving D. magna, over the test period, were as follows:

 

Parameter

Geometric Mean Measured Concentration of AEP (mg/L)

Control

0.003

0.006

0.019

0.163

0.324

Total Number of Offspring Produced*

1294

1477

1540

1362

957

1222

Mean Number of Offspring per SurvivingD. Magna*

67

78

77

76

64

61

* Note: values exclude immobileD. magnaobserved during the test period. 

 

Offspring from adults which were observed to be immobile during the reproduction period were omitted from the calculation of total number of offspring and mean number of offspring per D. magna. The first offspring were observed on Day 8 in the control, 0.003, 0.006 and 0.019 mg AEP/L treatment groups, reproduction was first observed on Day 9 in the 0.163 and 0.324 mg AEP/L treatment groups.

Statistical analysis was conducted to confirm the effect Savinase, Batch PPA 25057 had on D. magna reproduction. The EC5, EC10, EC20and EC50for reproduction at Day 14 were estimated to be 0.035, 0.052, 0.086 and 0.221 mg AEP/L, respectively (equivalent to 1.2, 1.7, 2.9 and 7.4 mg Savinase, Batch PPA 25057/L). The EC5, EC10, and EC20for reproduction at Day 21 were estimated to be 0.082, 0.145 and 0.292 mg AEP/L, respectively (equivalent 2.7, 4.8 and 9.7 mg Savinase, Batch PPA 25057/L). The EC50for reproduction at Day 21 could not be estimated due to low levels of inhibition. The NOEC for reproduction at Day 14 and Day 21 was 0.019 and 0.324 mg/L, respectively (equivalent to 0.6 and 10.8 mg Savinase, Batch PPA 25057/L).

 

During the test, test solution quality parameters were within the following ranges: pH: 7.33‑7.86; temperature: 19.3-20.8°C and dissolved oxygen concentration: 71.9-99.4% of air saturation value.

 

Validity criteria for a valid test were met: control mortality in the adult D. magnawas ≤20% (5%); the mean number of offspring produced per control adult was ≥60 (67) and the coefficient of variation around the mean number of offspring produced per control adult was ≤25% (6.79%).

Description of key information

After 21 days of exposure the no-observed effect concentration in the present study was 0.324 mg active enzyme protein/L  and the EC10 value was  0.145 mg active enzyme protein/L

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
0.145 mg/L

Additional information

Two Daphnia magna reproduction test have been performed according to OECD guideline 211, and in compliance with GLP. One test performed as a semi-static test with medium renewal every second day and one test in a flow-through system due to the very fast degradation/inactivation in the test media recorded in the semi-static study. Hence, only the results from the study applying the flow through system have been used for the assessment as exposure concentration could not be confirmed in the semi-static test system.