Registration Dossier

Administrative data

Endpoint:
fish short-term toxicity test on embryo and sac-fry stages
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-11-06 to 2013-11-18
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 212 (Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: viscous liquid
Details on test material:
- Name of test material (as cited in study report): Pentapropylensuccinic anhydride, reaction products with ethanolamine, sodium and triethanolamine
salts
- Substance type: UVCB
- Physical state: viscous liquid
- Appearance: brown
- Active matter: 100% (w/w)
- Purity test date: 2011-11-15
- Lot/batch No.: ESD0010887
- Expiration date of the lot/batch: 2013-12-13
- Stability under test conditions: not specified
- Stabilty in water: not specified
- Storage condition of test material: Room temperature, protected from light, in original container



Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations:
Nominal test concentrations: 2.50 – 5.00 – 10.0 – 20.0 – 40.0 mg/L

The test concentrations were based on the results of a preliminary range
finding test:

Egg Hatch / Hatching Time in the Preliminary Test (20 eggs / replicate):

Nominal test item Replicate Study day 3 Study day 4 Study day 5 Study day 6
concentration [mg/L] Egg Hatch [%]
50.0 1 0 --- --- ---
2 0 --- --- ---
Mean 0 --- --- ---
5.00 1 0 50 70 90
2 0 10 60 90
Mean 0 30 65 90
Control 1 10 80 95 95
2 0 55 85 95
Mean 5 68 90 95

--- = 100% Mortality, Removal of dead eggs/larvae



Overall Survival and Mortality in the Preliminary Test (40 eggs / concentration):

Nominal
test item concentration Survival [n] Post hatch survival [%] Overall survival [%] Overall mortality [%]
[mg/L]
Study day 3 Study day 4 Study day 5 Study day 8
50.0 0 0 0 0 0 0 100
5.00 37 37 37 36 100 90 10
Control 38 38 38 38 100 95 5


- Sampling method: The concentrations of the test item were analytically verified via LC-MS/MS from 3 sampling intervals at the start of the exposure
intervals and at the end of the exposure intervals of all concentration levels and the control by determination of the TIC of four analytes.

- Sample storage conditions before analysis: All original samples were stored at 6 ± 2 °C after stabilizing with methanol directly after sampling.
Prepared samples were stored in an autosampler at room temperature until analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 40 mg/L was freshly prepared with dilution water. The test concentrations of 2.50 to 20.0 mg/L were prepared from this
stock solution by dilution.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebrafish;
- Source: All fish used in the test were gained at DR.U.NOACK-LABORATORIEN from a single brood stock (supplier: Umweltbundesamt,
Schichauweg 58, 12307 Berlin, Germany)
- Method of breeding: A breeding stock of unexposed, mature zebrafish with an age of 12 months was used for egg production. Fish were free of
macroscopically discernable symptoms of infection and disease. Spawners were maintained in aquaria with a loading capacity of a minimum of
1 L water per fish.
- Temperature: 25 ± 2 °C
- Dissolved oxygen concentration > 60 % of air saturation value
- pH value: 6 – 8.5
- Photoperiod: 16 h light / 8 h dark cycle
(2 transition periods, 30 minutes each)
- Diffuse light (0.1 - 10 µmol photons * m-2 * s-1on water surface)
- Food: Artemia salina nauplii, 48 hours old, ad libitum;
dry food sera vipan SERA GMBH, ad libitum.
- No disease treatments were administered
- Tap water of local origin was used for holding. The water was filtered on activated charcoal and aerated for at least 24 h to remove
chlorine.
Nominal water parameters:
Total hardness: 10 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5

- Spawning: Adult zebrafish were kept in separate aquaria. About 15 minutes before start of artificial dawning rectangular dishes (26 cm x 14 cm x
6 cm), covered with a stainless steel mesh and provided with artificial plants, were introduced into the aquaria. After approximately 1 hour the
glass dishes were gently removed. A sufficient number of eggs was taken, washed in dilution water and immediately transferred into vessels
containing the respective exposure solutions without regard to fertilization (start of exposure). Eggs were fully covered with the respective test
solutions.

- Fertilization check: After approximately 2 h post fertilization, eggs were checked for fertilization. Under a stereo microscope every embryo was
checked for its blastomer phase. Eggs with only a 2 cell blastomer were regarded not to be fertilised. These eggs as well as coagulated eggs were
discarded.

- Introduction of eggs: Only fertilised eggs with more than 2 cells were introduced in the test vessels. 10 eggs were introduced per replicate
(corresponding to 30 eggs per treatment group).

- Feeding during test: no feeding was provided during the test. The sac-fry was nourished from the yolk-sac until end of exposure
(5 days post-hatch).

ACCLIMATION
- Acclimation not neccessary as breeding conditions are the same as test conditions

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
9 d
Post exposure observation period:
5 days post hatch

Test conditions

Hardness:
The total hardness, measured at the beginning of the exposure from one replicate per test concentration and control, was in the range of 55 – 71 mg CaCO3/L.
Test temperature:
The mean water temperature measured every renewal interval from freshly prepared and aged test solutions during exposure period in alternating replicates
of the control was 25.3 °C and ranged from a minimum temperature of 25.0 °C to a maximum temperature of 25.7 °C.

The water temperature was recorded continuously (once per hour) in a surrogate test chamber throughout exposure. The mean temperature +/- mean
standard deviation was 25.3 +/- 0.2 °C .

Water Temperature in the Test Media:

Study day Test media Water temperature [°C]
Nominal test item concentration [mg/L]
Control 2.50 5.00 10.0 20.0 40.0
0 new 25.2 25.2 25.1 25.1 25.0 25.1
2 old 25.3 25.2 25.6 25.0 25.1 25.1
new 25.7 25.0 25.4 25.4 25.7 -
5 old 25.3 25.0 25.4 25.3 25.5 -
new 25.3 25.2 25.1 25.2 - -
7 old 25.0 25.0 24.9 24.8 - -
new 25.1 25.2 25.2 25.1 - -
9 old 25.1 25.2 25.2 24.9 - -
Mean 25.3 25.1 25.2 25.1 25.3 25.1
SD ± 0.21 0.10 0.22 0.20 0.33 0.00
Min. 25.0 25.0 24.9 24.8 25.0 25.1
Max. 25.7 25.2 25.6 25.4 25.7 25.1


Water Temperature (Continuous Measuring) in the Dilution Water:

Period of measurements 2013-11-06 to 2013-11-15
Minimum temperature [°C] 24.8
Maximum temperature [°C] 25.6
Mean temperature ± Standard deviation [°C] 25.3 ± 0.2

pH:
The pH-values in the control and test item groups ranged from 7.12 to 7.54 and from 7.20 to 7.74 during exposure, respectively.


pH Values in the Test Media:

Study day Test media pH-value
Nominal test item concentration [mg/L]
Control 2.50 5.00 10.0 20.0 40.0
0 new 7.32 7.40 7.46 7.53 7.56 7.61
2 old 7.44 7.54 7.57 7.74 7.61 7.63
new 7.28 7.39 7.38 7.42 7.47 -
5 old 7.54 7.73 7.74 7.74 7.73 -
new 7.32 7.38 7.42 7.46 - -
7 old 7.12 7.35 7.41 7.47 - -
new 7.12 7.20 7.25 7.32 - -
9 old 7.41 7.48 7.57 7.57 - -
Mean 7.32 7.43 7.48 7.53 7.59 7.62
SD ± 0.15 0.16 0.15 0.15 0.11 0.01
Min. 7.12 7.20 7.25 7.32 7.47 7.61
Max. 7.54 7.73 7.74 7.74 7.73 7.63
Dissolved oxygen:
The dissolved oxygen concentrations in the control and test item groups, expressed in percent saturation, were in the range of 92 – 100 % during exposure.


Dissolved Oxygen in Percent Air Saturation Value:

Study day Test media Dissolved Oxygen [%]
Nominal test item concentration [mg/L]
Control 2.50 5.00 10.0 20.0 40.0
0 new 100 100 100 100 100 100
2 old 98 97 97 98 98 93
new 99 98 99 100 100 -
5 old 99 98 97 97 94 -
new 100 100 100 100 - -
7 old 97 97 97 94 - -
new 99 100 100 99 - -
9 old 97 96 95 92 - -
Mean 99 98 98 98 98 97
SD ± 1.19 1.58 1.89 3.02 2.83 4.95
Min. 97 96 95 92 94 93
Max. 100 100 100 100 100 100
Nominal and measured concentrations:
Nominal concentrations were 2.50 – 5.00 – 10.0 – 20.0 – 40.0 mg/L. The test concentrations were based on the results of a preliminary range finding test.
The measured concentrations of the test item at the start of the 1st exposure interval were in the range of 92 to 97 %, at the start of the 2nd exposure interval 94 to 97 % and at the start of the 3rd exposure interval 94 to 97 %. The measured concentrations of the test item at the end of the 1st exposure interval were in the range of 91 to 101 %, at the end of the 2nd exposure interval 104 to 111 % and at the end of the 3rd exposure interval 96 to 102 %.
For further details see "Any other information on materials and methods incl. tables".
Details on test conditions:
TEST SYSTEM
- Test vessel: Crystallisation dishes (inner diameter 13.5 cm, water height about 5 cm), Volume of the test media = approximately 500 mL
- Type (delete if not applicable): test vessel closed with a lid (with 1 hole, diameter approximately 1 cm)
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): semi-static test procedure with renewal of the test media every 2 to 3 days; for the renewal of
the test media the test organisms were retained in the test vessels whilst a proportion of at least 75 % of the test medium was changed. Freshly
prepared media was gently added to test vessels to avoid stressing of the test organisms.
- No. of organisms per vessel: 10 eggs per vessel
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin; the water was filtered on activated charcoal and aerated for at least 24 h to
remove chlorine; Total hardness: 10 - 250 mg CaCO3/L, pH-value: 6.0 - 8.5
- Total organic carbon: The total organic carbon of the dilution water (TOC, measured as NPOC) determined at the beginning of the exposure was
1.45 mg/L.
- Residual Chlorine: Residual chlorine of the dilution water, determined at the beginning of the exposure was ≤ 0.010 mg/L.
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature, pH value and oxygen saturation were measured on each water renewal interval from freshly
prepared and old test media in one replicate per test concentration and the control, respectively. Water temperature was recorded continuously by
a datalogger in a surrogate test vessel filled with dilution water. Total hardness was measured at the beginning of the exposure from one replicate
per test concentration and control, respectively. Chlorine and TOC were measured at the beginning of the test from the dilution water.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16/8 h photoperiod (light/dark)
- Light intensity: Light intensity was measured at start of exposure on the surface of the test vessels and ranged from 3.78 to 4.84 µmol photons *
m-2 * s-1 (mean: 4.29 µmol photons * m-2 *s-1). The test vessels were positioned randomly and repositioned daily.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Test concentrations: Nominal test concentrations: 2.50 – 5.00 – 10.0 – 20.0 – 40.0 mg/L
Reference substance (positive control):
not required

Results and discussion

Effect concentrations
Duration:
9 d
Dose descriptor:
NOEC
Effect conc.:
5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Hatch, overall survival, length
Details on results:
- Egg Fertilization Rate: the egg fertilization rate, determined on study day 0 (start of exposure) was > 97 %.
- Egg Hatch and Definition of Post Hatch Day 0: Egg hatch began on study day 3 in the control and the test concentrations of 2.50 and 5.00 mg/L.
Hatch continued until study day 4 (control and 2.50 mg/L) and study day 6 (5.00 mg/L). Study day 4 was determined to be post hatch day 0
(PHD 0) with a control hatching rate of 97 %. Egg hatch of the test concentration of 10.0 mg/L begun on study day 5 (PHD 1) and continued until
study day 7 (PHD 3). No hatch of eggs was observed for the test concentrations of 20.0 and 40.0 mg/L, since 100 % mortality occurred on study
5 and study day 2, respectively. Statistical procedures (One Way Analysis of Variance and DUNNETT’S Test) were applied for study days 3, 4, 5, 6
and 7 (post hatch days -1, 0, 1, 2 and 3). Statistically significant differences were found for study days 3, 4, 5 and 6.

Egg Hatch / Hatching Time:

Nominal
test item
concentration
[mg/L] Replicate Egg hatch [%]

PHD -2 PHD -1 PHD 0 PHD 1 PHD 2 PHD 3
(Study day 2) (Study day 3) (Study day 4) (Study day 5) (Study day 6) (Study day 7)
40.0 1 n.a. n.a. n.a. n.a. n.a. n.a.
2 n.a. n.a. n.a. n.a. n.a. n.a.
3 n.a. n.a. n.a. n.a. n.a. n.a.
Mean n.a. n.a. n.a. n.a. n.a. n.a.
20.0 1 0 0 0 n.a. n.a. n.a.
2 0 0 0 n.a. n.a. n.a.
3 0 0 0 n.a. n.a. n.a.
Mean 0 (+) 0 (+) 0 n.a. n.a. n.a.
10.0 1 0 0 0 10 80 90
2 0 0 0 10 60 60
3 0 0 0 0 70 80
Mean 0 (+) 0 (+) 0 (+) 7 (+) 70 (-) 77
5.00 1 0 10 90 90 100 100
2 0 10 90 90 100 100
3 0 0 90 100 100 100
Mean 0 (+) 7 (-) 90 (-) 93 (-) 100 (-) 100
2.50 1 0 50 100 100 100 100
2 0 60 100 100 100 100
3 0 20 90 90 90 90
Mean 0 (-) 43 (-) 97 (-) 97 (-) 97 (-) 97
Control 1 0 60 100 100 100 100
2 0 60 90 90 90 90
3 0 60 100 100 100 100
Mean 0 60 97 97 97 97

n.a. = Not applicable, due to 100 % mortality prior to hatch
(+) = Statistically significant difference from control (alpha = 0.05, ANOVA and DUNNETT’S Test)
(-) = No statistically significant difference from control (alpha = 0.05, ANOVA and DUNNETT’S Test)

- Swim-up: Swim-up was observed for a 3-day period on study days 5 to 7. Swim up of newly hatched fry of the control was observed for the first
time and completed on a single study day (study day 5; PHD 1). In the test concentrations of 2.50 and 5.00 mg/L newly hatched fry began to swim
up on study day 5 (post hatch day 1). The completion of swim-up in those test concentrations was on study 6 (PHD 2) and study day 7 (PHD 3),
respectively. For other tested concentrations the swim up behaviour could not be estimated, since mortality of eggs and behavioural effects of
newly hatched fry interfered with the observation of swim-up. No significance tests were carried out for swim-up.


Percent Swim-up of Hatched Fry:

Nominal test item
concentration
[mg/L] Replicate Swim-up [%]

PHD 0 PHD 1 PHD 2 PHD 3
(Study day 4) (Study day 5) (Study day 6) (Study day 7)
40.0 1 n.a. n.a. n.a. n.a.
2 n.a. n.a. n.a. n.a.
3 n.a. n.a. n.a. n.a.
Mean n.a. n.a. n.a. n.a.
20.0 1 0 n.a. n.a. n.a.
2 0 n.a. n.a. n.a.
3 0 n.a. n.a. n.a.
Mean 0 n.a. n.a. n.a.
10.0 1 0 0 0 n.d.
2 0 0 0 n.d.
3 0 0 0 n.d.
Mean 0 0 0 n.d.
5.00 1 0 56 90 100
2 0 89 90 100
3 0 70 100 100
Mean 0 72 93 100
2.50 1 0 100 100 100
2 0 70 100 100
3 0 78 100 100
Mean 0 83 100 100
Control 1 0 100 100 100
2 0 100 100 100
3 0 100 100 100
Mean 0 100 100 100

n.a. = Not applicable, due 100 % mortality prior to hatch
n.d. = Not determinable, since behavioural effects interfered with the swim up


- Fry Survival (Post Hatch Survival) : The post hatch success in all control replicates met the guideline criteria. The post hatch survival was
calculated from the study day with the maximum hatch rate (PHD 0: control and 2.50 mg/L; PHD 2: 5.00 mg/L; PHD 3: 10.0 mg/L) and the
surviving larvae on PHD 5. Post hatch survival for the test concentrations of 20.0 and 40.0 mg/L is not applicable, since 100 % mortality
occurred prior to hatch of eggs. The post hatch survival at the end of the study was 100 % in the control group and ranged from 66 to 100 % post
hatch success in the surviving test concentrations of 2.50 to 10.0 mg/L . One Way Analysis of Variance and DUNNETT’S Test were carried out for
post hatch success of surviving test concentrations at the end of the study. No statistically significant differences were found for the tested
concentrations when compared with the control.


Post Hatch Survival on Study Day 9 (Post Hatch Day 5):

Nominal
test item
concentration
[mg/L] Study day Replicate Hatched larvae on Vital larvae on Post hatch
with maximum hatch study day with study day 9 survival
hatch maximum hatch (PHD 5) [%]
n/10 n/10

40.0 n.a. 1 n.a. n.a. n.a.
2 n.a. n.a. n.a.
3 n.a. n.a. n.a.
Mean n.a. n.a. n.a.
20.0 n.a. 1 n.a. n.a. n.a.
2 n.a. n.a. n.a.
3 n.a. n.a. n.a.
Mean n.a. n.a. n.a.
10.0 7
(PHD 3) 1 9 7 78
2 6 2 33
3 8 7 88
Mean 8 5 (-) 66
5.00 6
(PHD 2) 1 10 10 100
2 10 10 100
3 10 10 100
Mean 10 10 (-) 100
2.50 4
(PHD 0) 1 10 10 100
2 10 10 100
3 9 9 100
Mean 10 10 (-) 100
Control 4
(PHD 0) 1 10 10 100
2 9 9 100
3 10 10 100
Mean 10 10 100

n.a. = Not applicable, due to 100 % mortality prior to hatch
(-) = No statistically significant difference from control (alpha = 0.05, ANOVA and DUNNETT’S Test)


- Overall Survival: Overall survival at the end of the study (PHD 5) was 97 % in the control group and ranged from 0 to 100 % in the tested
concentration levels. One Way Analysis of Variance and DUNNETT’S Test were carried out for the results of overall survival on PHD 5 (end of the
study). Statistically significant differences were found for the test concentrations of 10.0 to 40.0 mg/L when compared with the control.


Overall Survival and Mortality on Study Day 9 (Post Hatch Day 5):

Nominal
test item
concentration
[mg/L] Replicate Live fry on Overall survival Mortality
post hatch day 5 [%] [%]
(study day 9)
n/10

40.0 1 0 0 100
2 0 0 100
3 0 0 100
Mean 0 (+) 0 100
20.0 1 0 0 100
2 0 0 100
3 0 0 100
Mean 0 (+) 0 100
10.0 1 7 70 30
2 2 20 80
3 7 70 30
Mean 5 (+) 53 47
5.00 1 10 100 0
2 10 100 0
3 10 100 0
Mean 10 (-) 100 0
2.50 1 10 100 0
2 10 100 0
3 9 90 10
Mean 10 (-) 97 3
Control 1 10 100 0
2 9 90 10
3 10 100 0
Mean 10 97 3

(+) = Statistically significant difference from control (alpha = 0.05, ANOVA and DUNNETT’S Test)
(-) = No statistically significant difference from control (alpha = 0.05, ANOVA and DUNNETT’S Test)


- Fry Growth: The fry growth, expressed as length and dry weight, was determined on study day 9 (PHD 5; end of the study) from all surviving fish
larvae. One Way Analysis of Variance and DUNNETT’S Test were carried out for the results of PHD 5. For the length data of the concentration of
10.0 mg/L a statistically significant difference was found when compared with the control. For the weight data no statistically significant
differences were found for all surviving fish larvae of the test concentrations of 2.50 to 10.0 mg/L when compared with the control.
Fry Growth: Length and Dry Weight on Study Day 9 (Post Hatch Day 5):

Post hatch day 5 (study termination)

Nominal test item concentration Replicate Number Mean length Pooled Mean dry weight per concentration of per fish larvae dry weight fish larvae
[mg/L] larvae [mm] [mg] [mg]

10.0 1 7 3.786 0.420 0.047
2 2 3.743 0.344* 0.049*
3 7 3.898
Mean (+) 3.809 0.382 (-) 0.048
5.00 1 10 3.944 0.478 0.048
2 10 4.039 0.446 0.045
3 10 4.052 0.468 0.047
Mean (-) 4.012 0.464 (-) 0.047
2.50 1 10 4.091 0.474 0.047
2 10 4.106 0.430 0.043
3 9 4.122 0.454 0.050
Mean (-) 4.106 0.453 (-) 0.047
Control 1 10 4.120 0.488 0.049
2 9 4.098 0.428 0.048
3 10 4.024 0.444 0.044
Mean 4.081 0.453 0.047
SD ± 0.125 0.031 0.003
CV [%] 3.06 6.84 6.38

* fish larvae of replicate 2 and 3 were pooled for determination of dry weight

(+) = Statistically significant difference from control (alpha =0.05, ANOVA and DUNNETT’S Test)
(-) = No statistically significant difference from control (alpha =0.05, ANOVA and DUNNETT’S Test)

- Morphological and Behavioural Observations: from study days 7 to 9 (end of the study) larvae of the concentration level 10.0 mg/L showed
quiescence marked by abnormally low activity, as well as arresting on the ground. The same concentration level showed from study day 7 to 9 also
morphological abnormalities like scoliosis and oedema. Other larvae of the control group and the test concentrations of 2.50 and 5.00 mg/L
showed no morphological or behavioural abnormalities. Fisher’s Exact Test (two sided) was carried out for the observations of morphological and
behavioural abnormalities on study day 9 (PHD 5). For the non-lethal observations of the concentration level of 10.0 mg/L statistically significant
differences were found.

Morphological and Behavioural Observations:

Nominal Rep. Observation* Number of vital larvae affected at observation time
test item conc.
[mg/L]

PHD -1 PHD 0 PHD 1 PHD 2 PHD 3 PHD 4 PHD 5
(Study day 3) (Study day 4) (Study day 5) (Study day 6) (Study day 7) (Study day 8) (Study day 9)
40.0 1 - 3 (N) ---
20.0 1 - 3 (N) - - - ---
10.0 1 (N) - - 1/1 8/8 - - -
(A) - - - - 8/8 7/7 (+) 7/7
(Q) - - - - 8/8 7/7 (+) 7/7
(S) - - - - 8/8 7/7 (+) 7/7
(O) - - - - 8/8 7/7 (+) 7/7
2 (N) - - 1/1 6/6 - - -
(A) - - - - 5/5 4/4 (+) 2/2
(Q) - - - - 5/5 4/4 (+) 2/2
(S) - - - - 3/5 3/4 (+) 2/2
(O) - - - - 3/5 3/4 (+) 2/2
3 (N) - - - 7/7 - - -
(A) - - - - 7/7 7/7 (+) 7/7
(Q) - - - - 7/7 7/7 (+) 7/7
(S) - - - - 4/7 4/7 (+) 7/7
(O) - - - - 4/7 4/7 (+) 7/7
5.00 1 - 3 (N) All remaining- vital larvae
2.50 1 - 3 (N) All remaining- vital larvae
Control 1 - 3 (N) All remaining- vital larvae


(N) = Normal appearance and behaviour
(S) = Scoliosis (A) = Arresting on the ground
(O) = Oedema (Q) = Quiescence
- = No observations --- = No observation due to 100 % mortality

(+) = Statistically significant difference from control (Fisher’s Exact Test, two sided)












Reported statistics and error estimates:
One way analysis of variance (ANOVA) and DUNNETT’S Test was used for NOEC/LOEC calculations. When running a one way analysis of variance a normality
test and an equal variance test were done first. For the parameters hatch, post hatch survival and overall fry survival (mortality), growth (length and dry
weight), the following statistical tests were conducted: Hatching data of study days 3 to 7 (post hatch days -1 to 3) were analysed with ANOVA and DUNNETT’S
Test. Normality Tests failed for hatch data of study day 3, 4, and 5. No transformation was carried out with these data because the data set was not estimated tofollow a normal distribution. Post hatch survival and overall survival data (mortality), dry weight and length data of study day 9 (post hatch day 5) were
analysed with ANOVA. DUNNETT’S Test was used for NOEC/LOEC calculation of overall survival data and length data of study day 9. Normality Test failed for
post hatch survival data and overall survival data. No transformation was carried out with these data because the data set was not estimated to follow a normal distribution. Morphological effects (scoliosis, oedema) and behavioural effects (quiescence and arresting on the ground), observed on study day 9, were
analysed with Fisher’s exact test (two sided), as revised in OECD Series on Testing and Assessment No. 54 (2006). The statistical analyses were conducted with conclusions of statistical significance based on a 95 % confidence level. The alpha-value (acceptable probability of incorrectly concluding that there is a
difference) was 0.05.

The LC50-value and the corresponding confidence interval after 5 days post-hatch were calculated by sigmoidal dose-response regression analysis
(0 to 100 regression) as revised in OECD Series on Testing and Assessment No. 54 (2006).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Pentapropylensuccinic anhydride, reaction products with ethanolamine, sodium and triethanolamine salts caused significant effects on the short-term toxicity with embryo and sac-fry stages of zebrafish under semi-static conditions at the dosage levels of 10.0 to 40.0 mg/L (nominal test item concentration).
The LC50 on study day 9 (post hatch day 5) was determined to be 10.1 (5.89 – 17.4) mg/L. The NOEC for the parameter hatch was 5.00 mg/L. The NOEC for
the parameters overall survival (mortality) and length growth was 5.00 mg/L. The NOEC for the parameters post hatch survival and dry weight was 10.0 mg/L.
All effect levels were based on the nominal concentrations of Pentapropylensuccinic anhydride, reaction products with ethanolamine, sodium and
triethanolamine salts.
Executive summary:

The effects of the test item Pentapropylensuccinic anhydride, reaction products with ethanolamine, sodium and triethanolamine salts (batch no.:ESD0010887) to the embryo and sac-fry stages of fish (Zebrafish /Danio rerio) were determined according to OECD Guideline 212 from 2013‑11‑06 to 2013-11-18, with the definitive exposure phase from 2013 -11 -06 to 2013 -11-15 at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany.

A semi-static test procedure with renewal of the test media every 2 to 3 days was performed with the nominal test item concentrations of

2.50 – 5.00 – 10.0 – 20.0 – 40.0 mg/L (factor 2.0).

The test was started by placing fertilized eggs in the test vessels and lasted 9 days (5 days post-hatch). 30 eggs of Danio reriowere exposed per test concentration and control (3 replicates with 10 eggs each), respectively.

 

On day four 97 % of the control larvae have hatched. Therefore, study day 4 was defined as post hatch day 0 (= PHD 0).

 

Different toxic endpoints were determined: egg hatch, time to hatch, post hatch survival, overall fry survival, mortality and fry growth (expressed as length and dry weight), respectively. The results of the named parameters were checked for statistically significant differences. The NOEC, LOEC and LC-values were determined based on the statistical results.

 

The analytical monitoring of Pentapropylensuccinic anhydride, reaction products with ethanolamine, sodium and triethanolamine salts in the fresh test media (study days 0, 2 and 5) and in the corresponding aged test media (study days 2, 5 and 7) of various concentration levels and the control was carried out via LC-MS/MS analysis.

The measured concentrations of the test item Pentapropylensuccinic anhydride, reaction products with ethanolamine, sodium and triethanolamine salts in the freshly prepared test media were in the range of 92 to 97 % of the nominal values. The measured concentrations of the test item in the corresponding aged test media were in the range of 91 to 111 % ofthe nominal values.

All effect levels were based on the nominal concentrations of the test item Pentapropylensuccinic anhydride, reaction products with ethanolamine, sodium and triethanolamine salts.

NOEC, LOEC: Hatch, Fry Survival, Fry Growth and Observations on Abnormal Morphology and Behaviour

[based on nominal test item concentrations [mg/L]

Parameter

NOEC

LOEC

Hatch

  5.00

   10.0

Post hatch survival

10.0

> 10.0

Overall survival

  5.00

   10.0

Length

  5.00

   10.0

Dry weight

10.0

> 10.0

Morphological observations (scoliosis and oedema)

  5.00

   10.0

Behavioural observations (arresting on the ground and quiescence)

  5.00

   10.0

LC50-Value with 95 % Confidence Interval on Study Day 9 (Post Hatch Day 5)

                [based on nominal test item concentrations [mg/L]

 

LC50

95 % confidence interval

10.1

5.89 – 17.4