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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 25 to April 30, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
see below
Vehicle:
no
Details on test solutions:
see below
Test organisms (species):
Oryzias latipes
Details on test organisms:
see below
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Details on test conditions:
see below
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
0.82 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
values based on mean measured concentrations calculated directly from data in the study report.
Basis for effect:
mortality
Remarks on result:
other: 95% CL
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.526 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
Concentrations of PAP maintained between 56.6 an d 61.5 % depending on the nominal concentration
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
0.809 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95%CL
Details on results:
Concentrations of PAP maintained between 56.6 an d 61.5 % depending on the nominal concentration. Values based on mean measured concentrations calculated directly from data in the study report.

- Measured Concentrations: The test concentrations were measured at the start and the end of the test using HPLC.

Nominal Conc. (mg/L) Measured Conc. (mg/L) (% of Nominal) Mean* Measured Conc. (mg/L)
0 hr (new) 96hr (old)
Control 0.0220 0.0220 0.0220
0.365 0.214(54.1) 0.272(68.9) 0.243(61.5)
0.593 0.278(46.8) 0.401(67.6) 0.339(57.2)
0.889 0.500(56.2) 0.553(62.2) 0.526(59.2)
1.33 0.765(57.5) 0.854(64.2) 0.809(60.8)
2 1.09(54.5) 1.17(58.7) 1.13(56.6)

*: Arithmetic mean of measured concentrations


- Water chemistry (pH, DO and temperature in test): Water chemistry and temperature were measured for each concentration at least once a day.
pH: 7.3 - 7.6
DO: 7.9 - 8.3 mg/L (more than 60% of saturation)
Water Temperature: 23.0 - 24.2 C

-Effect Data(mortality):
LC50 (96hr) 0. 82 mg/L (mc)
95% Confidence interval: 0.780 - 1.11 mg/L
NOEC (96hr) 0.526 mg/L (mc)
mc: based on measured concentrations

- Cumulative Mortality: None of test organisms were killed during exposure period at control, 0.243, 0.339 and 0.526 mg/L.

Measured Conc. (mg/L) Cumulative Number of Dead (Percent Mortality)
24 hour 48 hour 72 hour 96 hour
Control 0 (0) 0 (0) 0 (0) 0 (0)
0.243 0 (0) 0 (0) 0 (0) 0 (0)
0.339 0 (0) 0 (0) 0 (0) 0 (0)
0.526 0 (0) 0 (0) 0 (0) 0 (0)
0.809 0 (0) 3 (30) 3 (30) 3 (30)
1.13 2 (20) 7 (70) 8 (80) 8 (80)



-Other Effect:
As toxic symptoms, abnormal swimming behaviour and immobility were observed during test period in concentrations of 0.526, 0.809 and 1.13 mg/L. No symptoms were observed in control, 0.243 and 0.339 mg/L.

Appearance of test solutions in concentrations of 1.13 and 0.809 mg/L were slightly brown and transparent, while that of other concentrations were colourless and transparent.


Validity criteria fulfilled:
yes
Conclusions:
96 hour LC50 is 0.82 mg/L based on measured concentrations.
Endpoint:
fish embryo acute toxicity (FET)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From October to November 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
The purpose of this non GLP study was to determine the effects of the test item (parent molecule) and its possible degradation products on the development of embryos of zebra fish (Danio rerio).
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 236 (Fish embryo acute toxicity (FET) test)
Deviations:
yes
Remarks:
4 concentration levels, no analytical monitoring.
Principles of method if other than guideline:
- Principle of test: The purpose of this non GLP study was to determine the effects of the test item (parent molecule) and its possible degradation products on the development of embryos of zebra fish (Danio rerio). In order to compare the toxic potential of the parent molecule with the toxic potential of its possible degradation products, the study comprised two experimental parts: In Experimental Part A, the parent molecule of the test item was tested. In Experimental Part B, the degradation products of the test item were tested.
- Short description of test conditions: For Experimental Part A, a semi-static test design with test medium renewal after 24 hours was applied. Experimental Part B was performed static. The exposure periods of both experimental parts were run in parallel. The duration of the test was 96 hours.
For each treatment, 20 eggs were introduced into the test for each of the two experimental parts.
The test item nominal concentrations for both experimental parts were 0.10, 1.0, 10, and 100 mg/L. For each experimental part, a control (test water without test item) was run in parallel.
- Parameters analysed / observed: The eggs/embryos were observed daily for coagulated embryos, lack of somite formation, non-detachment of the tail, mortality and hatching of larvae.
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I80561
- Expiration date of the lot/batch: 2 June 2017
- Purity test date: 3 June 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the fridge, protect from light and moisture or heat

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
In order to compare the toxic potential of the parent molecule and its possible degradation products, two different ways of dosing were chosen:
Experimental Part A (Parent Molecule): The test medium of the highest nominal test concentration of 100 mg/L was prepared at the start of the test and at the test medium renewals at Day 1, 2, and 3 by mixing nominal 100 mg (effective range: 100.1 to 100.0) of the test item into 1000 mL test water using intense stirring for 10 minutes at room temperature in the dark. The freshly prepared test medium of the highest test concentration was diluted with test water to prepare the test media of the lower test concentrations. Application and setup of the test was carried out as quickly as possible in order to avoid degradation of the test substance as far as possible.
Experimental Part B (Degradation Products): The test medium of the highest nominal concentration of 100 mg/L was prepared by mixing 100.3 mg the test item into 1000 mL test water using intense stirring for 96 hours at room temperature in the dark. It was assumed, that during the long stirring time, the parent molecule was degraded to its degradation products. After stirring, the aged test medium of the highest test concentration was diluted with test water to prepare the test media of the lower test concentrations.

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No, the appearance of the test media was recorded daily; see below for the results
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: danio rerio
- Age at study initiation (mean and range, SD): The study was performed with freshly fertilized eggs of zebra fish (Danio rerio). At the start of the test, the eggs were introduced into the test media approximately 2 hours after fertilization.
Test type:
other: Experiment A: semi-static, Experiment B: static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
Dilution water: 125 mg/L as CaCO3
Test temperature:
The test was performed under temperature-controlled conditions (water bath). The water temperature in the water bath was constantly 26°C.
pH:
The pH values were measured at the start and the end of the test medium renewals (Experimental Part A) and at the start and the end of the test (Experimental Part B) in all test media and the controls and were in the range of 7.6 to 7.8.
Dissolved oxygen:
The oxygen concentration was measured in the freshly prepared test media and was in the range of 8.3 to 8.6 mg/L.
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal: 0.1-1.0-10-100 mg/L, for experimental parts A and B
Details on test conditions:
TEST SYSTEM
- Test vessel: Polystyrene multi well plates with approximately 3 mL filling capacity per well
- Type (delete if not applicable): open
- Aeration: The test vessels were not aerated during the test
- Type of flow-through (e.g. peristaltic or proportional diluter): not appropriate
- Renewal rate of test solution (frequency/flow rate): not appropriate
- No. of organisms per vessel: 1 egg per well
- No. of vessels per concentration (replicates): 20 eggs per plate/concentration
- No. of vessels per control (replicates): 20 eggs
- No. of vessels per vehicle control (replicates): not appropriate

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water was used in the study. It consisted of analytical grade salts dissolved in purified water to obtain the following nominal concentrations: CaCl2 × 2H2O: 147 mg/L, MgSO4 × 7H2O: 61.5 mg/L, NaHCO3: 32.5 mg/L, KCl: 2.9 mg/L
- Alkalinity: 0.4 mmol/L
- Intervals of water quality measurement: The pH values were measured at the start and the end of the test medium renewals
(Experimental Part A) and at the start and the end of the test (Experimental Part B. For measurements at the end of the exposure, the contents of all wells per treatment was combined.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: A 16-hour light to 8-hour dark cycle with a 30 minutes transition period was used. The light conditions were slightly reduced by covering the water bath with white paper folia.
- Light intensity: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The eggs/embryos were observed daily for coagulated embryos, lack of somite formation, non-detachment of the tail, mortality and hatching of larvae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline: 4 concentration levels, non GLP study
- Range finding study: no
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Experimental part A (parent molecule)
Key result
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Experimental part A (parent molecule)
Key result
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
0.15 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Experimental part A (parent molecule)
Remarks:
95% Conf. Limits not determined
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Experimental part B (degradation products)
Key result
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Experimental part B (degradation products)
Key result
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Experimental part B (degradation products)
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Experimental part B (degradation products)
Remarks:
95% Conf. Limits not determined
Details on results:
- Behavioural abnormalities: no
- Observations on body length and weight: not appropriate
- Other biological observations: All surviving larvae showed a normal appearance and no visible abnormalities were observed during their development regarding somite formation or non-detachment of the tail.
- Mortality of control: 2 dead eggs in exp A and 2 dead eggs in exp B
- Other adverse effects control: no
- Abnormal responses: not reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not appropriate, but apperance of the test media was recorded (see below for details)
- Effect concentrations exceeding solubility of substance in test medium: not appropriate since the test item is highly soluble.
Reported statistics and error estimates:
The LC50 and LC100 values for larvae mortality were estimated by a Probit Analysis. The NOEC and LOEC were determined directly from the raw data.

Effect of test item on survival of larvae of Zebra Fish: Experimental part A (parent molecule)

 
       Larvae hatched/viable eggs  N° of larvae hatched  Dead eggs  Hatching rate (%)  % of control
   day 1  day 2  day 3  day 4        
 Control 0/18   0/18  1/17 18/0   18 2 90   --
 0.10  0/16  0/16  0/16 16//0  16 80  89 
 1.0  0/0  0/0  0/0  0/0  0 20 
 10  0/0  0/0  0/0  0/0  0 20 
 100  0/0  0/0  0/0  0/0  0 20 

Effect of test item on survival of larvae of Zebra Fish: Experimental part B (degradation products):

 
       Larvae hatched/viable eggs  N° of larvae hatched  Dead eggs  Hatching rate (%)  % of control
   day 1  day 2  day 3  day 4        
 Control 0/19   0/19  2/17 18/0   18 2 90   --
 0.10  0/16  0/15  1/14 15//0  15 75  83 
 1.0  0/17  0/17  6/11 16/0  16 80  89 
 10  0/0  0/0  0/0  0/0  0 20 
 100  0/0  0/0  0/0  0/0  0 20 

Validity criteria: overall survival of embryos in the negative (dilution-water) control was ≥ 90% until the end of the 96 hrs exposure; Hatching rate in the negative control was ≥ 80% at the end of 96 hrs exposure; The water temperature in the water bath was constantly 26°C.

Conclusions:
The results obtained during these 96-hour toxicity tests indicate, that the parent molecule of the test item is more toxic to larvae of zebra fish compared to its degradation products.
Executive summary:

The purpose of this non GLP study based on OECD 236, was to determine the effects of the test item (parent molecule) and its possible degradation products on the development of embryos of zebra fish (Danio rerio). In order to compare the toxic potentials, the study comprised two experimental parts: In Experimental Part A, the parent molecule of the test item was tested. In Experimental Part B, the degradation products of the test item were tested. For Experimental Part A, a semi-static test design with test medium renewal after 24 hours was applied. Experimental Part B was performed static.

The exposure periods of both experimental parts were run in parallel. The duration of the test was 96 hours. For each treatment, 20 eggs were introduced into the test for each of the two experimental parts. The nominal test item concentrations for both experimental parts were 0.10, 1.0, 10, and 100 mg/L. For each experimental part, a control (test water without test item) was run in parallel. The eggs/embryos were observed daily for coagulated embryos, lack of somite formation, non detachment of the tail, mortality and hatching of larvae. The appearance of the test media were recorded daily. No analytical work was performed.

For experimental part A (parent molecule), LC50 -96H=0.15 mg/L, LC100=1.0 mg/L, NOEC=0.10 mg/L, LOEC=1.0 mg/L.

For experimental part B (degradation products), LC50 -96H=1.4 mg/L, LC100=10 mg/L, NOEC=1.0 mg/L, LOEC=10 mg/L.

The results obtained during these 96-hour toxicity tests indicate, that the parent molecule of the test item is more toxic to larvae of zebra fish compared to its degradation products.

Description of key information

The short-term 96 hr LC50 for fish, based on the mean of measured concentrations, was 0.82 mg/L in Oryzias latipes as reported in the key study. The NOEC value was 0.526mg/L.

In addition to the acute toxicity study a fish embryo acute toxicity (FET) study was conducted on both the parent and degradation product. The results of this study found that the parent product was more toxic than the degradation products.

parent molecule, LC50 -96H=0.15 mg/L, LC100=1.0 mg/L, NOEC=0.10 mg/L, LOEC=1.0 mg/L

degradation products, LC50 -96H=1.4 mg/L, LC100=10 mg/L, NOEC=1.0 mg/L, LOEC=10 mg/L.

Key value for chemical safety assessment

LC50 for freshwater fish:
0.82 mg/L

Additional information

The key study is an Acute Toxicity in Oryzias latipes completed in 1997 based on OECD method 203 and GLP with a reported LC50 based on mean of measured concentration of LC50 of 0.82 mg/L. Concentrations of PAP maintained between 56.6 and 61.5 % depending on the nominal concentration.Values based on mean measured concentrations calculated directly from data in the study report.