Registration Dossier
Registration Dossier
Data platform availability banner - registered substances factsheets
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Diss Factsheets
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 700-342-7 | CAS number: 1163775-81-2
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Long-term toxicity to fish
Administrative data
Link to relevant study record(s)
- Endpoint:
- fish early-life stage toxicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 26/01/2022 - 02/03/2022
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- A read-across justification is provided in Section 13.
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
- Version / remarks:
- (2013)
- Principles of method if other than guideline:
- N/A
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- N/A
- Analytical monitoring:
- yes
- Details on sampling:
- Sampling schedule
Samples of test media including control group and solvent control group were taken from alternating test replicates and the mixing chamber supply of these replicates on study days -1, 0, and weekly thereafter until end of exposure. The changing intervals of the stock solution were considered.
Sampling and pre-treatment
At each sampling day 2 samples were taken per (alternating) test replicate. For each sample at least 30 mL of each test item concentration, the solvent control and the control were sampled.
For analysis of fresh and aged stock solutions, approx. 3 mL of each stock solution were sampled. - Vehicle:
- yes
- Remarks:
- With regard to the limited solubility of the test item in water, ethanol (VWR, 99.97%, batch 21DO014013) was used as a solvent. The solvent concentration was the same in all concentration levels and the solvent control (0.025 mL/L).
- Details on test solutions:
- Stock solution
A stock solution of 1120 mg/L was prepared in ethanol. An appropriate amount of the test item was pipetted into a glass flask and filled up with the solvent. Density of the test item was taken into account. The solution was agitated until it was visually clear dissolved.
The stock solutions were prepared in appropriate intervals of 7 days.
Syringes were filled with the freshly prepared stock solutions or pure ethanol for the solvent control in corresponding intervals.
Details are given in Table 3 (attached).
Control
Dilution water (without test item and without solvent).
Solvent control
A solvent control with the same concentration of solvent but without test item was prepared and tested under the same conditions as the test groups. - Test organisms (species):
- Danio rerio (previous name: Brachydanio rerio)
- Details on test organisms:
- Origin
Fish were bred at the test facility from a single brood stock (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin, Germany)
Maintenance of brood fish
A breeding stock of unexposed, mature zebrafish aged between 9 and 11 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.
Spawning
15 – 35 adult zebrafish were kept in at least 3 separate aquaria. The fish were healthy with a mortality rate < 5% during the last 7 days and thus not medically treated for at least 7 days. About 15 minutes before artificial dawn, rectangular dishes covered with a stainless-steel mesh and containing artificial plants (plastic), were introduced into the aquaria. After 1 hour the glass dishes were removed. Any unhealthy eggs, as well as coagulated and un-fertilized eggs, were discarded (less than 30%). About 900 eggs were taken and washed in dilution water. Eggs originated from 3 different spawnings. - Test type:
- flow-through
- Water media type:
- freshwater
- Remarks:
- 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.
- Limit test:
- yes
- Total exposure duration:
- 35 d
- Remarks on exposure duration:
- (30 days post-hatch)
- Post exposure observation period:
- N/A
- Hardness:
- 10 – 250 mg CaCO3/L
- Test temperature:
- 26 ± 1.5 °C
- pH:
- 6.0 – 8.5
- Dissolved oxygen:
- The dissolved oxygen concentrations in the control, solvent control and the test item groups, expressed in percent saturation, were in the mean 94 - 96% and ranged from 84 to 100% during the exposure period (see Table 13 and Table 14).
- Salinity:
- N/A
- Conductivity:
- 172 µS/cm (recent measurement: 2021-11-09)
- Nominal and measured concentrations:
- The test was performed with three nominal test concentrations of 7.00 - 14.0 - 28.0 µg test item/L, which corresponded to arithmetric mean measured concentrations 4.72 - 8.45 - 13.9 µg/L of the dispersed fraction of the test item.
- Details on test conditions:
- Test design
A randomized block design with each treatment being present in each block was established. A flow-through exposure design was carried out. Membrane piston pumps provided the water flow-through. Precision syringe pumps were used for the introduction of stock solution. The stock solution and the dilution water were mixed in a mixing chamber (approx. volume 0.7 L) by magnetic stirring before passing the test aquaria (approx. volume 7.5 L; four replicates per test concentration, control and solvent control) where the eggs/fish were exposed. Glass tubes were used.
The accuracy of the water flow-through was checked prior to start of the exposure and three times per week thereafter. Water exchange in the test aquaria was about 10 times per day (3.125 L/h).
An equilibration period of at least 3 days was carried out prior to start of the exposure until measured concentrations of the test item showed no trend of increasing or decreasing levels.
Equilibration period
Test solutions flowed through the test vessels for 22 days prior to the start of the exposure until measured concentrations of the test item no trend of increasing or decreasing.
Control
Dilution water (without test item and without solvent)
Solvent control
Additionally, a solvent control with the same concentration of solvent but without test item was prepared and tested under the same conditions as the test groups.
Test duration
35 days (30 days post hatch), depending on post-hatch day 0 (study day 5).
Replicates, number of eggs
Four replicates per test concentration and control, with 20 eggs each (80 eggs per test concentration, solvent control and control) were tested.
For the whole study (including the range finding test and definitive test) 467 fish were used.
Loading
A loading rate not exceeding 0.5 g/L wet weight fish per 24 hours and not exceeding 5 g/L of solution at any time was maintained.
Test vessels
Glass aquaria of 8.7 L provided with mesh coated fittings allowing flow-through of test media (dimensions: 22/22/18 cm) were used. Test vessels were covered by glass lids. The volume of the test media was approximately 7.5 L.
Cleaning
The test vessels were siphoned as needed to remove excess fecal material and uneaten food, and to minimize microbial growth and biodegradation of the test item.
The mesh coated fittings were cleaned daily after start of feeding. Cleaning started on study day 5.
Aeration
The dilution water supply tank was aerated.
No additional aeration was provided.
Feeding of test fish
The feeding regime was ad libitum during the whole feeding period (study day 5 to 34).
Feeding started 3 days after the beginning of hatch on study day 5 (post-hatch day 1, where almost all non-affected larvae swum up). Larvae were fed with starter food (ST-1 (AQUA SCHWARZ GMBH, 37081 Göttingen, Germany), as well as a suspension of the starter food ST-1 and fine milled brine shrimp nauplii (2 – 8 times daily). 1 day after start of feeding brine shrimp nauplii (48 h old) were fed until the end of the test (4 – 7 times daily).
Light intensity (target) and photoperiod
300 ± 150 Lux
A daily 16 / 8 h photoperiod (light / dark) was maintained throughout exposure. - Reference substance (positive control):
- no
- Remarks:
- No reference item is recommended for this test according to the guideline.
- Key result
- Duration:
- 6 d
- Dose descriptor:
- NOEC
- Effect conc.:
- >= 13.9 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat. (dissolved fraction)
- Basis for effect:
- number hatched
- Key result
- Duration:
- 30 d
- Dose descriptor:
- NOEC
- Effect conc.:
- >= 13.9 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat. (dissolved fraction)
- Basis for effect:
- length
- Key result
- Duration:
- 30 d
- Dose descriptor:
- NOEC
- Effect conc.:
- >= 13.9 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat. (dissolved fraction)
- Basis for effect:
- weight
- Key result
- Duration:
- 30 d
- Dose descriptor:
- NOEC
- Effect conc.:
- >= 13.9 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat. (dissolved fraction)
- Basis for effect:
- mortality
- Remarks:
- Post-hatch survival
- Key result
- Duration:
- 30 d
- Dose descriptor:
- NOEC
- Effect conc.:
- >= 13.9 µg/L
- Nominal / measured:
- meas. (arithm. mean)
- Conc. based on:
- test mat. (dissolved fraction)
- Basis for effect:
- mortality
- Remarks:
- Overall survival
- Key result
- Duration:
- 6 d
- Dose descriptor:
- NOEC
- Effect conc.:
- >= 28 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat. (total fraction)
- Basis for effect:
- number hatched
- Key result
- Duration:
- 30 d
- Dose descriptor:
- NOEC
- Effect conc.:
- >= 28 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat. (total fraction)
- Basis for effect:
- length
- Key result
- Duration:
- 30 d
- Dose descriptor:
- NOEC
- Effect conc.:
- >= 28 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat. (total fraction)
- Basis for effect:
- weight
- Key result
- Duration:
- 30 d
- Dose descriptor:
- NOEC
- Effect conc.:
- >= 28 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat. (total fraction)
- Basis for effect:
- mortality
- Remarks:
- Post-hatch survival
- Key result
- Duration:
- 30 d
- Dose descriptor:
- NOEC
- Effect conc.:
- >= 28 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat. (total fraction)
- Basis for effect:
- mortality
- Remarks:
- Total survival
- Reported statistics and error estimates:
- Statistical analysis (NOEC) of hatching success (study days 5 & 6)
Comparison between Control and Solvent control for Hatching Success at Study Day 5 (PHD 0)
Shapiro-Wilk´s Test on Normal Distribution
Treatment [µg/L] Mean s n
Control 1.35 0.000 4
Solvent 1.51 0.113 4
Results:
Number of residuals = 7; Shapiro-Wilk´s W = 0.968; p(W) = 0.703; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.
Normality check was passed (Shapiro-Wilk´s; p > 0.01).
The F test revealed significantly different variances (p <= 0.01).
STUDENT-t test for Homogeneous Variances
Treatm. [µg/L] Mean s df %MDD t p(t) Sign. p(F)
Control 1.35 0.000
Solvent 1.51 0.113 6 10.3 3.00 0.024 + n.d.
+: significant; -: non-significant - Validity criteria fulfilled:
- yes
- Remarks:
- Dissolved oxygen saturation - between 84 and 100%. Water temperature was in the recommended range. Hatching success was 96% in the control and 99% in the solvent control groups. Post-hatch survival was 79% in control and 84% in solvent control groups.
- Conclusions:
- 1, 2, 4-Benzenetricarboxylic acid, mixed decyl and octyl triesters (CAS-No. 90218-76-1) caused no significant effects on Zebrafish in an early life stage test, 30 days post hatch when tested with nominal concentrations of 7.00, 14.0 and 28.0 µg/L.
For the parameter hatching success, the nominal NOEC was determined to be ≥ 28.0 µg/L. Corresponding to the arithmetric mean measured concentration NOEC of ≥ 13.9 µg/L of the dispersed fraction of the test item.
For the parameters post hatch survival and overall survival, the nominal NOECs were determined to be ≥ 28.0 µg/L, corresponding to the arithmetric mean measured NOEC of ≥ 13.9 µg/L of the dispersed fraction of the test item.
For the parameter fry growth (expressed as length and fresh weight) the nominal NOECs were ≥ 28.0 µg/L (for both parameters). Corresponding to the arithmetric mean measured concentration NOECs of ≥ 13.9 µg/L of the dispersed fraction of the test item.
All effect values are given based on the nominal test item concentrations and the arithmetric mean measured concentrations of the dispersed fraction of the test item 1, 2, 4-Benzenetricarboxylic acid, mixed decyl and octyl triesters (CAS-No. 90218-76-1). - Executive summary:
The effects of the test item 1, 2, 4 Benzenetricarboxylic acid, mixed decyl and octyl triesters (CAS-No. 90218-76-1) (Batch-No. 05804/MA) on the early-life stage of fish (Danio rerio / Zebrafish) were determined according to OECD Guideline 210.
Stock solutions in ethanol with nominal concentrations of 280, 560 and 1120 mg/L were prepared at intervals of 7 days and continuously dosed in a flow-through system. Based on the results of a range finding test, a dose-response test was conducted with nominal test item concentrations 7.00, 14.0 and 28.0 µg/L, corresponding to the arithmetric mean measured concentrations 4.72 - 8.45 - 13.9 µg/L of the dispersed fraction of the test item.
The test lasted 35 days (30 days post-hatch). 80 eggs of Danio rerio / zebrafish were exposed to each test concentration, the solvent control and the control (4 replicates with 20 eggs each).
Water quality parameters pH-value, oxygen concentration, temperature and total hardness were within the acceptable limits.
On study day 5, 95% of the control and 99% the solvent control larvae had hatched. Therefore, study day 5 was defined as post hatch day 0 (= PHD 0).
The following toxicological endpoints were determined: hatching success, fry growth (assessed via length and fresh weight measurements on PHD 30), morphological and behavioral effects, post-hatch survival and overall survival.
Concentrations of the test item and controls were determined during exposure on day 0, day 7, day 14, day 21 and day 28 for all tested concentration levels and for three constituents C8, C8, C10-BTE, C8, C10, C10 BTE and C10, C10, C10-BTE via LC MS/MS.
1, 2, 4-Benzenetricarboxylic acid, mixed decyl and octyl triesters (CAS-No. 90218-76-1) caused no significant effects on Zebrafish in an early life stage test, 30 days post hatch when tested with nominal concentrations of 7.00, 14.0 and 28.0 µg/L.
For all parameters measured, the nominal NOEC was determined to be ≥ 28.0 µg/L. Corresponding to the arithmetric mean measured concentration NOEC of ≥ 13.9 µg/L of the dispersed fraction of the test item.
Reference
Biological Results
Egg Fertilization Rate
The mean egg fertilization rate determined on study day 0 (start of the
exposure) was 92%.
Eggs were fully covered with the respective test solutions during
fertilization check.
Hatch and Definition of Post Hatch Day 0
Hatch began on study day 2 in the test concentration of 28.0 µg/L. The hatch of larvae in other test concentrations and control groups started on study day 3. The hatch of larvae was completed until study day 6. Study day 5 was determined to be post hatch day 0 (PHD 0) with a hatching rate of 95% in the control and 99% in the solvent control (see Table 4).
Statistical procedures were applied for the total number of test
organisms that have hatched on study days 5 and 6.
The William’s multiple t-test procedure (alpha = 0.05) for hatch data
after 5 and 6 days a significance level of 0.05 was applied. No
statistically significant differences were found between the pooled
controls and the nominal test concentrations of 7.00 to 28.0 µg/L.
The NOEC and the LOEC (based on nominal test item concentrations) for
this endpoint were determined to be ≥ 28.0 and > 28.0 µg/L,
corresponding to the arithmetric mean measured concentrations of ≥ 13.9
and > 13.9 µg/L of the dispersed fraction of the test item, respectively.
Swim-up
The swim-up period of the control groups and the nominal test concentrations of 7.00 to 28.0 µg/L was observed from study day 5 to 6. First swim-up of larvae was observed on study day 5 in all test groups. For details see Table 5. No statistical analysis of swim-up data was carried out.
Fry Survival (Post-Hatch Survival)
The post-hatch survival in the control replicates met the validity
criteria of the guideline (required: ≥ 75%). The fry survival
(post-hatch survival) at the end of the study was 79% in the control and
84% in the solvent control, thus fully meeting the validity criteria of
the guideline given in part 8. No concentration-related decrease of the
post-hatch survival was detected with increasing test concentrations.
The post-hatch survival data of the test concentrations are given in
Table 6.
Dunnett’s multiple t-test was performed on post-hatch survival data on
study day 35 (PHD 30). No statistically significant differences were
found between the pooled control groups and the nominal test item
concentrations of 7.00 to 28.0 µg/L.
The NOEC and the LOEC for this endpoint were ≥ 28.0 µg/L and > 28.0 µg/L
(nominal concentrations), corresponding to the arithmetric mean measured
concentrations of ≥ 13.9 and > 13.9 µg/L of the dispersed fraction of
the test item, respectively.
Overall Survival
The cumulative mortality at the end of the exposure, related to the
number of eggs introduced on day 0, was 24% for the control and 17% for
the solvent control. No concentration-related decrease of the overall
survival (increase of overall mortality) was detected in the test item
concentrations (see Table 7).
The Dunnett’s multiple t-test was performed for statistical analysis of
overall survival data on study day 35 (PHD 30). No statistically
significant differences were found between the pooled control groups and
the nominal test item concentrations of 7.00 to 28.0 µg/L.
The NOEC and the LOEC for this endpoint were ≥ 28.0 µg/L and > 28.0 µg/L
(nominal concentrations), corresponding to the arithmetric mean measured
concentrations of ≥ 13.9 and > 13.9 µg/L of the dispersed fraction of
the test item, respectively.
Morphological and Behavioral Effects
Observation of abnormal appearance and behavior of hatched larvae were
carried out daily until the end of the exposure. No morphological and no
biological significant behavioral effects were observed in the control,
solvent control and in the nominal test concentrations of 7.00 to 28.0
µg/L. The observed behavioural effects were very isolated events of
single larvae during exposure.
On study day 8 and 24 one larvae of the control group showed an unusual
long arresting on the ground and quiescence.
On study day 11 and 12 one larvae of the solvent control group showed
tumbling. On study days 21 to 24 one to two larvae showed an unusual
long arresting on the ground and quiescence, respectively.
On study day 6 and 7 two larvae of the lowest test concentration (7.00
µg/L) showed an unusual long arresting on the ground. Tumbling and a
slowed escape reflex was also observed for these larvae on study days 7
to 10, respectively.
On study day 24 one larvae on the middle concentration (14.0 µg/L) group
showed an unusual long arresting on the ground and quiescence.
On study day 11, 22 to 24 one to two larvae of the highest concentration
(28.0 µg/L) group showed an unusual long arresting on the ground and
quiescence.
Fry Growth
Fry growth, expressed as length and wet weight measurements, was
measured on study day 35 (PHD 30) from all survivors (see Table 8 and
Table 9). For the individual length data, refer to Table 10 and Table
11. Pooled wet weights based on replicate means are given in Table 12.
The Dunnett’s multiple t-test showed no statistically significant
differences for the surviving fish of the nominal test concentrations of
7.00 to 28.0 µg/L, respectively, for both growth parameters (mean total
length and fresh weight). Therefore, the NOEC and the LOEC (nominal test
item concentrations) for the growth parameter total length were
determined to be ≥ 28.0 and > 28.0 µg/L, corresponding to the
arithmetric mean measured concentrations of ≥ 13.9 and > 13.9 µg/L of
the dispersed fraction of the test item, respectively.
Biomass Loading
The biomass-loading factor for the study was determined from the fresh weights of the control and solvent control fish at the end of the exposure (see Table 12).
The maximum biomass at the end of the exposure was determined in replicate 1 of the solvent control group: 711.0 mg total fish weight. The maximum biomass loading based on the 7.5 liter volume of a single growth chamber was 94.8 mg/L.
Maximum loading rate: biomass / volume of test solution = 711.0 mg / 7.5 L = 94.8 mg/L
The biomass loading rate based upon a flow of 75 liters per day through each single test aquaria was 9.48 mg per liter and day.
Maximum loading rate per day: biomass / volume of test solution per day = 711.0 mg / 75 L = 9.48 mg/L per day.
These loadings were well within the requirements to ensure adequate dissolved oxygen levels and to avoid crowding of the fish.
Reported statistics
Statistical Analysis of Hatching Success
Statistical Significance (NOEC) – Hatching Success (Study Days 5 and 6)
Comparison between Control and Solvent control for Hatching Success at Study Day 5 (PHD 0)
Shapiro-Wilk´s Test on Normal Distribution
Shapiro-Wilk´s Test on Normal Distribution with hatchability at 5 d:
Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability
of the W statistic (i.e. that the observed deviations from the normal
distributions are dues to chance). In case p(ShapiroWilk´s W) is greater
than the chosen significance level, the normality hypothesis(Ho) is
accepted.
Treatm. [µg/L] Mean s n
Control 1.35 0.000 4
Solvent 1.51 0.113 4
Results:
Number of residuals = 7; Shapiro-Wilk´s W = 0.968; p(W) = 0.703; p(W) is
greater than the selected significance level of 0.010; thus treatment
data do not significantly deviate from normal distribution.
Normality check was passed (Shapiro-Wilk´s; p > 0.01).
The F test revealed siginificantly different variances (p <= 0.01).
STUDENT-t test for Homogeneous Variances
STUDENT-t test for Homogeneous Variances with hatchability at 5 d: Two-sample comparison of the two controls. Significance was Alpha = 0.050, two-sided; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Control (in percent of Control); t: sample t; p(t): probability of sample t for Ho: µ1 = µ2; the differences are significant in case p(t) <= Alpha ; p(F): two-sided probability of F computed by the F-test (Ho: var1 = var2 (homogeneity); p(F) > 0.010 is the criterion of variance homogeneity. (Control(c) and treatment(t) variance was applied: s²(c)/nc + s²(t)/nt, each).
Treatm. [µg/L] Mean s df %MDD t p(t) Sign. p(F)
Control 1.35 0.000
Solvent 1.51 0.113 6 10.3 3.00 0.024 + n.d.
+: significant; -: non-significant
A significant difference between Control and each treatment was shown. The effect was considered not to be biological. Therefore, control and solvent control samples were pooled.
Comparison between Control and Solvent control for Hatching Success at Study Day 6
Shapiro-Wilk´s Test on Normal Distribution
Shapiro-Wilk´s Test on Normal Distribution with hatchability at 6 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level, the normality hypothesis(Ho) is accepted.
Treatm. [µg/L] Mean s n
Control 1.40 0.113 4
Solvent 1.51 0.113 4
Results:
Number of residuals = 6; Shapiro-Wilk´s W = 0.954; p(W) = 0.629; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.
Normality check passed (Shapiro-Wilk´s; p > 0.01).
Variance homogeneity check (F-test) passed (p > 0.01).
STUDENT-t test for Homogeneous Variances
STUDENT-t test for Homogeneous Variances with hatchability at 6 d: Two-sample comparison of the two controls. Significance was Alpha = 0.050, two-sided; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Control (in percent of Control); t: sample t; p(t): probability of sample t for Ho: µ1 = µ2; the differences are significant in case p(t) <= Alpha ; p(F): two-sided probability of F computed by the F-test (Ho: var1 = var2 (homogeneity); p(F) > 0,010 is the criterion of variance homogeneity. (Control(c) and treatment(t) variance was applied: s²(c)/nc + s²(t)/nt, each).
Treatm. [µg/L] Mean s df %MDD t p(t) Sign. p(F)
Control 1.40 0.113
Solvent 1.51 0.113 6 13.9 1.41 0.207 - 1.000
+: significant; -: non-significant
There is no statistically significant difference between control and solvent.
Threshold concentrations (NOEC) for Hatching Success at Study Day 5 (PHD 0)
Shapiro-Wilk´s Test on Normal Distribution
Shapiro-Wilk´s Test on Normal Distribution with hatchability at 5 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level, the normality hypothesis(Ho) is accepted.
Treatm. [µg/L] Mean s n
Pooled Control 1.43 0.117 8
7.000 1.51 0.113 4
14.000 1.49 0.161 4
28.000 1.40 0.113 4
Results:
Number of residuals = 8; Shapiro-Wilk´s W = 0.946; p(W) = 0.613; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.
Levene´s Test on Variance Homogeneity (with Residuals)
Levene´s Test on Variance Homogeneity (with Residuals) with hatchability at 5 d: Source: source of variance; SS: sum of squares; df: degrees of freedom; MSS: mean sum of squares; F: test statistic: p: probability that the variance explained by the treatment is due to chance
Source SS df MSS F p(F)
Treatment 0.0039 3 0.0013 0.465 0.711
Residuals 0.0444 16 0.0028
Total 0.048 19
The Levene test indicates variance homogeneity (p > 0.010).
Variance homogeneity check was passed (p > 0.01).
Normal-distribution and variance-homogeneity requirements are fulfilled and so the user-selected multiple test was performed.
To justify the use of Williams test at first a trend analysis by contrasts was performed.
Trend analysis by Contrasts (Monotonicity of Concentration/Response)
Trend analysis by contrasts (monotonicity of concentration/response) with hatchability at 5 d: Psi: sum of means weighted by contrasts; s(psi): standard error of psi; df: degrees of freedom; t: t-statistic; p(t): probability that the trend is due to chance (Ho: Slope = 0). Hypothesis of monotonicity is accepted if at least the linear contrast is significant.
Trend Psi s(psi) df t p(t)
Linear 0,1086 0,2457 16 0,442 0,332
Quadratic 0,1733 0,1168 16 1,484 0,079
The linear trend was not significant (p > 0,05) The quadratic trend was not significant (p > 0,05)
The analysis of contrasts did not reveal a linear trend, nonetheless the user-selected Williams test was performed.
Williams Multiple Sequential t-test Procedure
Comparison of treatments with "Pooled Control" by the t test procedure after Williams with hatchability at 5 d: Significance was Alpha = 0.050, one-sided smaller; Mean: arithmetic mean; n: sample size; s: standard deviation; LhM: max. likelihood mean; MDD: minimum detectable difference to Pooled Control (in percent of Pooled Control); t: sample t; 't*: critical t for Ho: µ1 = µ2 = ... = µk; the differences are significant in case |t| > |t*| (The residual variance of an ANOVA was applied; df = N - k; N: sum of treatment replicates n(i); k: number of treatments). Note that the step-down test terminates after the first non-significant treatment is encountered
Treatm. [µg/L] Mean s df LhM %MDD t t* Sign.
Pooled Control 1.43 0.1248
7.000 1.51 0.1248 16 1.51 -9.3 1.11 -1.75 -
14.000 1.49 0.1248 16 1.49 -9.7 0.79 -1.82 -
28.000 1.40 0.1248 16 1.40 -9.8 -0.37 -1.84 -
+: significant; -: non-significant
The NOEC appears to be higher than or equal 28.0 µg/L.
Threshold concentrations (NOEC) for Hatching Success at Study Day 6
Shapiro-Wilk´s Test on Normal Distribution
Shapiro-Wilk´s Test on Normal Distribution with hatchability at 6 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level, the normality hypothesis(Ho) was accepted.
Treatm. [µg/L] Mean s n
Pooled Control 1.46 0.121 8
7.000 1.51 0.113 4
14.000 1.49 0.161 4
28.000 1.40 0.113 4
Results:
Number of residuals = 8; Shapiro-Wilk´s W = 0.948; p(W) = 0.620; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.
Levene´s Test on Variance Homogeneity (with Residuals)
Levene´s Test on Variance Homogeneity (with Residuals) with hatchability at 6 d: Source: source of variance; SS: sum of squares; df: degrees of freedom; MSS: mean sum of squares; F: test statistic: p: probability that the variance explained by the treatment is due to chance
Source SS df MSS F p(F)
Treatment 0.0047 3 0.0016 0.655 0.591
Residuals 0.0385 16 0.0024
Total 0.043 19
The Levene test indicated variance homogeneity (p > 0.010).
Variance homogeneity check was passed (p > 0.01).
Normal-distribution and variance-homogeneity requirements were fulfilled, and so parametric multiple test was appropriate.
To justify the use of Williams test at first a trend analysis by contrasts was performed.
Trend analysis by Contrasts (Monotonicity of Concentration/Response)
Trend analysis by contrasts (monotonicity of concentration/response) with hatchability at 6 d: Psi: sum of means weighted by contrasts; s(psi): standard error of psi; df: degrees of freedom; t: t-statistic; p(t): probability that the trend is due to chance (Ho: Slope = 0). Hypothesis of monotonicity is accepted if at least the linear contrast is significant.
Trend Psi s(psi) df t p(t)
Linear 0.1932 0.2488 16 0.776 0.224
Quadratic 0.1451 0.1182 16 1.227 0.119
The linear trend is not significant (p > 0.05) The quadratic trend is not significant (p > 0.05)
The analysis of contrasts did not reveal a linear trend, thus the selected Williams test was replaced by Dunnett test.
Dunnett`s Multiple t-test Procedure
Dunnett´s multiple t-test procedure with hatchability at 6 d: Comparison of treatments with "Pooled Control". Significance was Alpha = 0,050, one-sided smaller (multiple level); Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Pooled Control (in percent of Pooled Control); t: sample t; t*: critical t for Ho: µ1 = µ2 = ... = µk; the differences are significant in case |t| > |t*| (The residual variance of an ANOVA was applied; df = N - k; N: sum of treatment replicates n(i); k: number of treatments).
Treatm. [µg/L] Mean s df %MDD t t* Sign.
Pooled Control 1.46 0.1264
7.000 1.51 0.1264 16 -12.1 0.73 -2.27 -
14.000 1.49 0.1264 16 -12.1 0.42 -2.27 -
28.000 1.40 0.1264 16 -12.1 -0.73 -2.27 -
+: significant; -: non-significant
The NOEC appears to be higher than or equal 28.0 µg/L.
Statistical Analysis of Post-Hatch Survival
Statistical Significance (NOEC) – Post-Hatch Survival on Study Day 35 (PHD 30)
Comparison between Control and Solvent control for Post-hatch Survival at Study Day 35
Shapiro-Wilk´s Test on Normal Distribution
Shapiro-Wilk´s Test on Normal Distribution with post-hatch survival at 35 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level, the normality hypothesis(Ho) is accepted.
Treatm. [µg/L] Mean s n
Control 1.11 0.177 4
Solvent 1.17 0.145 4
Results:
Number of residuals = 18; Shapiro-Wilk´s W = 0.958; p(W) = 0.557; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.
Normality check was passed (Shapiro-Wilk´s; p > 0.01).
Variance homogeneity ckeck (F-test) was passed (p > 0.01).
STUDENT-t test for Homogeneous Variances
STUDENT-t test for Homogeneous Variances with post-hatch survival at 35 d: Two-sample comparison of the two controls. Significance was Alpha = 0.050, two-sided; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Control (in percent of Control); t: sample t; p(t): probability of sample t for Ho: µ1 = µ2; the differences are significant in case p(t) <= Alpha ; p(F): two-sided probability of F computed by the F-test (Ho: var1 = var2 (homogeneity); p(F) > 0,010 is the criterion of variance homogeneity. (Control(c) and treatment(t) variance was applied: s²(c)/nc + s²(t)/nt, each).
Treatm. [µg/L] Mean s df %MDD t p(t) Sign. p(F)
Control 1.11 0.177
Solvent 1.17 0.145 6 25.1 0.48 0.649 - 0.747
+: significant; -: non-significant
There was no statistically significant difference between control and solvent.
Threshold concentrations (NOEC) for Post-Hatch Survival
Shapiro-Wilk´s Test on Normal Distribution
Shapiro-Wilk´s Test on Normal Distribution with post-hatch survival at 35 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level. the normality hypothesis(Ho) is accepted.
Treatm. [µg/L] Mean s n
Pooled Control 1.14 0.153 8
7.000 1.07 0.126 4
14.000 1.19 0.043 4
28.000 1.25 0.217 4
Results:
Number of residuals = 18; Shapiro-Wilk´s W = 0.967; p(W) = 0.734; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.
Levene´s Test on Variance Homogeneity (with Residuals)
Levene´s Test on Variance Homogeneity (with Residuals) with post-hatch survival at 35 d: Source: source of variance; SS: sum of squares; df: degrees of freedom; MSS: mean sum of squares; F: test statistic: p: probability that the variance explained by the treatment is due to chance
Source SS df MSS F p(F)
Treatment 0.0353 3 0.0118 1.688 0.210
Residuals 0.1115 16 0.0070
Total 0.147 19
The Levene test indicates variance homogeneity (p > 0.010).
Variance homogeneity check was passed (p > 0.01).
Normal-distribution and variance-homogeneity requirements are fulfilled.
A parametric multiple test was suitable.
To justify the use of Williams test at first a trend analysis by contrasts is performed.
Trend analysis by Contrasts (Monotonicity of Concentration/Response)
Trend analysis by contrasts (monotonicity of concentration/response) with post-hatch survival at 35 d: Psi: sum of means weighted by contrasts; s(psi): standard error of psi; df: degrees of freedom; t: t-statistic; p(t): probability that the trend is due to chance (Ho: Slope = 0). Hypothesis of monotonicity is accepted if at least the linear contrast is significant.
Trend Psi s(psi) df t p(t)
Linear 0.4495 0.2941 16 1.528 0.073
Quadratic 0.1388 0.1398 16 0.993 0.168
The linear trend was not significant (p > 0.05) The quadratic trend is not significant (p > 0.05)
The analysis of contrasts did not reveal a linear trend, thus the selected Williams test was replaced by Dunnett test.
Dunnett`s Multiple t-test Procedure
Dunnett´s multiple t-test procedure with post-hatch survival at 35 d: Comparison of treatments with "Pooled Control". Significance was Alpha = 0.050, one-sided smaller (multiple level); Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Pooled Control (in percent of Pooled Control); t: sample t; t*: critical t for Ho: µ1 = µ2 = ... = µk; the differences are significant in case |t| > |t*| (The residual variance of an ANOVA was applied; df = N - k; N: sum of treatment replicates n(i); k: number of treatments).
Treatm. [µg/L] Mean s df %MDD t t* Sign.
Pooled Control 1.14 0.1494
7.000 1.07 0.1494 16 -18.2 -0.82 -2.27 -
14.000 1.19 0.1494 16 -18.2 0.50 -2.27 -
28.000 1.25 0.1494 16 -18.2 1.20 -2.27 -
+: significant; -: non-significant
The NOEC appears to be higher than or equal 28.0 µg/L.
Statistical Analysis of Overall Survival
Statistical
Significance (NOEC) – Overall Survival Study Day 35 (PHD 30)
Comparison
between Control and Solvent control for Overall Survival
Shapiro-Wilk´s Test on Normal Distribution
Shapiro-Wilk´s Test on Normal Distribution with surival at 35 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level, the normality hypothesis(Ho) is accepted.
Treatm. [µg/L] Mean s n
Control 1.07 0.161 4
Solvent 1.15 0.148 4
Results:
Number of residuals = 18; Shapiro-Wilk´s W = 0.965; p(W) = 0.700; p(W) is greater than the selected significance level of 0.010; thus treatment data did not significantly deviate from normal distribution.
Normality check was passed (Shapiro-Wilk´s; p > 0.01).
Variance homogeneity check (F-test) was passed (p > 0.01).
STUDENT-t test for Homogeneous Variances
STUDENT-t test for Homogeneous Variances with surival at 35 d: Two-sample comparison of the two controls. Significance was Alpha = 0.050, two-sided; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Control (in percent of Control); t: sample t; p(t): probability of sample t for Ho: µ1 = µ2; the differences are significant in case p(t) <= Alpha ; p(F): two-sided probability of F computed by the F-test (Ho: var1 = var2 (homogeneity); p(F) > 0,010 is the criterion of variance homogeneity. (Control(c) and treatment(t) variance was applied: s²(c)/nc + s²(t)/nt, each).
Treatm. [µg/L] Mean s df %MDD t p(t) Sign. p(F)
Control 1.07 0.161
Solvent 1.15 0.148 6 24.9 0.75 0.482 - 0.894
+: significant; -: non-significant
There was no statistically significant difference between control and solvent.
Threshold concentrations (NOEC) for Overall Survival
Shapiro-Wilk´s Test on Normal Distribution
Shapiro-Wilk´s Test on Normal Distribution with surival at 35 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level, the normality hypothesis(Ho) is accepted.
Treatm. [µg/L] Mean s n
Pooled Control 1.11 0.150 8
7.000 1.06 0.136 4
14.000 1.16 0.084 4
28.000 1.17 0.129 4
Results:
Number of residuals = 16; Shapiro-Wilk´s W = 0.960; p(W) = 0.660; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.
Levene´s Test on Variance Homogeneity (with Residuals)
Levene´s Test on Variance Homogeneity (with Residuals) with surival at 35 d: Source: source of variance; SS: sum of squares; df: degrees of freedom; MSS: mean sum of squares; F: test statistic: p: probability that the variance explained by the treatment is due to chance
Source SS df MSS F p(F)
Treatment 0.0059 3 0.0020 0.276 0.842
Residuals 0.1151 16 0.0072
Total 0.121 19
The Levene test indicates variance homogeneity (p > 0.010).
Variance homogeneity check was passed (p > 0.01).
Normal-distribution and variance-homogeneity requirements are fulfilled.
A parametric multiple test is advisable.
To justify the use of Williams test at first a trend analysis by contrasts is performed.
Trend analysis by Contrasts (Monotonicity of Concentration/Response)
Trend analysis by contrasts (monotonicity of concentration/response) with surival at 35 d: Psi: sum of means weighted by contrasts; s(psi): standard error of psi; df: degrees of freedom; t: t-statistic; p(t): probability that the trend is due to chance (Ho: Slope = 0). Hypothesis of monotonicity is accepted if at least the linear contrast is significant.
Trend Psi s(psi) df t p(t)
Linear 0.2693 0.2618 16 1.029 0.160
Quadratic 0.0645 0.1244 16 0.518 0.306
The linear trend is not significant (p > 0.05) The quadratic trend is not significant (p > 0.05)
The analysis of contrasts did not reveal a linear trend. thus the selected Williams test was replaced by Dunnett test.
Dunnett`s Multiple t-test Procedure
Dunnett´s multiple t-test procedure with surival at 35 d: Comparison of treatments with "Pooled Control". Significance was Alpha = 0.050. one-sided smaller (multiple level); Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Pooled Control (in percent of Pooled Control); t: sample t; t*: critical t for Ho: µ1 = µ2 = ... = µk; the differences are significant in case |t| > |t*| (The residual variance of an ANOVA was applied; df = N - k; N: sum of treatment replicates n(i); k: number of treatments).
Treatm. [µg/L] Mean s df %MDD t t* Sign.
Pooled Control 1.11 0.1330
7.000 1.06 0.1330 16 -16.6 -0.70 -2.27 -
14.000 1.16 0.1330 16 -16.6 0.58 -2.27 -
28.000 1.17 0.1330 16 -16.6 0.68 -2.27 -
+: significant; -: non-significant
The NOEC appears to be higher than or equal 28.0 µg/L.
Statistical Analysis of Fry Growth – Fresh Weight
Statistical Significance (NOEC) – Fry Growth: Fresh Weight on Study Day 35 (PHD 30)
Comparison between Control and Solvent control for Fresh Weight
Shapiro-Wilk´s Test on Normal Distribution
Shapiro-Wilk´s Test on Normal Distribution with fresh weight at 35 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level, the normality hypothesis(Ho) is accepted.
Treatm. [µg/L] Mean s n
Control 39.750 6.7501 4
Solvent 38.025 3.8257 4
Results:
Number of residuals = 19; Shapiro-Wilk´s W = 0.941; p(W) = 0.278; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.
Normality check was passed (Shapiro-Wilk´s; p > 0.01)
Variance homogeneity ckeck (F-test) was passed (p > 0.01).
STUDENT-t test for Homogeneous Variances
STUDENT-t test for Homogeneous Variances with fresh weight at 35 d: Two-sample comparison of the two controls. Significance was Alpha = 0.050. two-sided; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Control (in percent of Control); t: sample t; p(t): probability of sample t for Ho: µ1 = µ2; the differences are significant in case p(t) <= Alpha ; p(F): two-sided probability of F computed by the F-test (Ho: var1 = var2 (homogeneity); p(F) > 0.010 is the criterion of variance homogeneity. (Control(c) and treatment(t) variance was applied: s²(c)/nc + s²(t)/nt. each).
Treatm. [µg/L] Mean s df %MDD t p(t) Sign. p(F)
Control 39.750 6.7501
Solvent 38.025 3.8257 6 23.9 -0.44 0.672 - 0.376
+: significant; -: non-significant
There is no statistically significant difference between control and solvent.
Threshold concentrations (NOEC) for Fresh Weight
Shapiro-Wilk´s Test on Normal Distribution
Shapiro-Wilk´s Test on Normal Distribution with fresh weight at 35 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level, the normality hypothesis(Ho) is accepted.
Treatm. [µg/L] Mean s n
Pooled Control 38.888 5.1623 8
7.000 41.575 3.4023 4
14.000 34.425 2.9239 4
28.000 34.250 2.0273 4
Results:
Number of residuals = 19; Shapiro-Wilk´s W = 0.930; p(W) = 0.173; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.
Levene´s Test on Variance Homogeneity (with Residuals)
Levene´s Test on Variance Homogeneity (with Residuals) with fresh weight at 35 d: Source: source of variance; SS: sum of squares; df: degrees of freedom; MSS: mean sum of squares; F: test statistic: p: probability that the variance explained by the treatment is due to chance
Source SS df MSS F p(F)
Treatment 13.72290 3 4.57430 0.737 0.545
Residuals 99.32552 16 6.20784
Total 113.0484 19
The Levene test indicates variance homogeneity (p > 0.010).
Variance homogeneity check was passed (p > 0.01).
Normal-distribution and variance-homogeneity requirements are fulfilled.
The user-selected multiple test is performed, according to the statistical guidance (annex5) of the test guideline OECD 210.
Dunnett`s Multiple t-test Procedure
Dunnett´s multiple t-test procedure with fresh weight at 35 d: Comparison of treatments with "Pooled Control". Significance was Alpha = 0.050. one-sided smaller (multiple level); Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Pooled Control (in percent of Pooled Control); t: sample t; t*: critical t for Ho: µ1 = µ2 = ... = µk; the differences are significant in case |t| > |t*| (The residual variance of an ANOVA was applied; df = N - k; N: sum of treatment replicates n(i); k: number of treatments).
Treatm. [µg/L] Mean s df %MDD t t* Sign.
Pooled Control 38.888 4.02534
7.000 41.575 4.02534 16 -14.4 1.09 -2.27 -
14.000 34.425 4.02534 16 -14.4 -1.81 -2.27 -
28.000 34.250 4.02534 16 -14.4 -1.88 -2.27 -
+: significant; -: non-significant
The NOEC appears to be higher than or equal 28.0 µg/L.
Statistical Analysis of Fry Growth – Mean Total Length
Statistical
Significance (NOEC) – Fry Growth: Mean Total Length on Study Day 35 (PHD
30)
Comparison
between Control and Solvent control for Mean Total Length
Shapiro-Wilk´s Test on Normal Distribution
Shapiro-Wilk´s Test on Normal Distribution with length at 35 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level, the normality hypothesis(Ho) is accepted.
Treatm. [µg/L] Mean s n
Control 15.85 0.968 4
Solvent 15.40 0.762 4
Results:
Number of residuals = 19; Shapiro-Wilk´s W = 0.931; p(W) = 0.184; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.
Normality check was passed (Shapiro-Wilk´s; p > 0.01).
Variance homogeneity ckeck (F-test) was passed (p > 0.01).
STUDENT-t test for Homogeneous Variances
STUDENT-t test for Homogeneous Variances with length at 35 d: Two-sample comparison of the two controls. Significance was Alpha = 0.050, two-sided; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Control (in percent of Control); t: sample t; p(t): probability of sample t for Ho: µ1 = µ2; the differences are significant in case p(t) <= Alpha ; p(F): two-sided probability of F computed by the F-test (Ho: var1 = var2 (homogeneity); p(F) > 0,010 is the criterion of variance homogeneity. (Control(c) and treatment(t) variance was applied: s²(c)/nc + s²(t)/nt, each).
Treatm. [µg/L] Mean s df %MDD t p(t) Sign. p(F)
Control 15.85 0.968
Solvent 15.40 0.762 6 9.5 -0.73 0.492 - 0.703
+: significant; -: non-significant
There is no statistically significant difference between control and solvent.
Threshold concentrations (NOEC) for Mean Total Length
Shapiro-Wilk´s Test on Normal Distribution
Shapiro-Wilk´s Test on Normal Distribution with length at 35 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level, the normality hypothesis(Ho) is accepted.
Treatm. [µg/L] Mean s n
Pooled Control 15.62 0.841 8
7.000 15.82 0.741 4
14.000 15.40 0.548 4
28.000 15.17 0.403 4
Results:
Number of residuals = 18; Shapiro-Wilk´s W = 0.963; p(W) = 0.663; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.
Levene´s Test on Variance Homogeneity (with Residuals)
Levene´s Test on Variance Homogeneity (with Residuals) with length at 35 d: Source: source of variance; SS: sum of squares; df: degrees of freedom; MSS: mean sum of squares; F: test statistic: p: probability that the variance explained by the treatment is due to chance
Source SS df MSS F p(F)
Treatment 0.2857 3 0.0952 0.585 0.633
Residuals 2.6037 16 0.1627
Total 2.889 19
The Levene test indicates variance homogeneity (p > 0.01).
Variance homogeneity check was passed (p > 0.01)
Normal-distribution and variance-homogeneity requirements are fulfilled.
A parametric multiple test is advisable.
To justify the use of Williams test at first a trend analysis by contrasts is performed.
Trend analysis by Contrasts (Monotonicity of Concentration/Response)
Trend analysis by contrasts (monotonicity of concentration/response) with length at 35 d: Psi: sum of means weighted by contrasts; s(psi): standard error of psi; df: degrees of freedom; t: t-statistic; p(t): probability that the trend is due to chance (Ho: Slope = 0). Hypothesis of monotonicity is accepted if at least the linear contrast is significant.
Trend Psi s(psi) df t p(t)
Linear 1.7750 1.3911 16 1.276 0.110
Quadratic 0.4250 0.6610 16 0.643 0.265
The linear trend is not significant (p > 0.05) The quadratic trend is not significant (p > 0.05)
The analysis of contrasts did not reveal a linear trend, thus the selected Williams test was replaced by Dunnett test.
Dunnett`s Multiple t-test Procedure
Dunnett´s multiple t-test procedure with length at 35 d: Comparison of treatments with "Pooled Control". Significance was Alpha = 0.050, one-sided smaller (multiple level); Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Pooled Control (in percent of Pooled Control); t: sample t; t*: critical t for Ho: µ1 = µ2 = ... = µk; the differences are significant in case |t| > |t*| (The residual variance of an ANOVA was applied; df = N - k; N: sum of treatment replicates n(i); k: number of treatments).
Treatm. [µg/L] Mean s df %MDD t t* Sign.
Pooled Control 15.62 0.7067
7.000 15.82 0.7067 16 -6.3 0.46 -2.27 -
14.000 15.40 0.7067 16 -6.3 -0.52 -2.27 -
28.000 15.17 0.7067 16 -6.3 -1.04 -2.27 -
+: significant; -: non-significant
The NOEC appears to be higher than or equal 28.0 µg/L.
Description of key information
The effects of the structurally similar read-across test item 1, 2, 4 Benzenetricarboxylic acid, mixed decyl and octyl triesters (CAS-No. 90218-76-1) (Batch-No. 05804/MA) on the early-life stage of fish (Danio rerio / Zebrafish) were determined according to OECD Guideline 210. 1, 2, 4-Benzenetricarboxylic acid, mixed decyl and octyl triesters (CAS-No. 90218-76-1) caused no significant effects on Zebrafish in an early life stage test, 30 days post hatch when tested with nominal concentrations of 7.00, 14.0 and 28.0 µg/L. For all parameters measured, the nominal NOEC was determined to be ≥ 28.0 µg/L. Corresponding to the arithmetric mean measured concentration NOEC of ≥ 13.9 µg/L of the dispersed fraction of the test item. The test results showed that the NOEC for the test substance to Danio rerio was higher than the concentration of the saturated solution under the test conditions. No toxicity was observed under test conditions in the range of water solubility.
Under the tested conditions, the threshold level of lethal effect for Gobiocypris rarus exposed to the test substance 1,2,4-Benzenetricarboxylic acid in 14 d was higher than the concentration of the saturated solution (measured concentration was below the LOD, LOD=0.274 mg/L). The threshold level of growth rate inhibition was higher than the concentration of the saturated solution (measured concentration was below the LOD, LOD=0.274 mg/L) and the no observed effect concentration was equal or greater than the saturated solution (measured concentration was below the LOD, LOD=0.274 mg/L). The test results showed that the NOEC for the test substance to Gobiocypris rarus was higher than the concentration of the saturated solution under the test conditions. No toxicity was observed under test conditions in the range of water solubility.
Key value for chemical safety assessment
Fresh water fish
Fresh water fish
- Dose descriptor:
- NOEC
- Effect concentration:
- >= 13.9 µg/L
Additional information
The effects of the test item 1, 2, 4 Benzenetricarboxylic acid, mixed decyl and octyl triesters (CAS-No. 90218-76-1) (Batch-No. 05804/MA) on the early-life stage of fish (Danio rerio / Zebrafish) were determined according to OECD Guideline 210.
Stock solutions in ethanol with nominal concentrations of 280, 560 and 1120 mg/L were prepared at intervals of 7 days and continuously dosed in a flow-through system. Based on the results of a range finding test, a dose-response test was conducted with nominal test item concentrations 7.00, 14.0 and 28.0 µg/L, corresponding to the arithmetric mean measured concentrations 4.72 - 8.45 - 13.9 µg/L of the dispersed fraction of the test item. The test lasted 35 days (30 days post-hatch). 80 eggs of Danio rerio / zebrafish were exposed to each test concentration, the solvent control and the control (4 replicates with 20 eggs each). Water quality parameters pH-value, oxygen concentration, temperature and total hardness were within the acceptable limits.
On study day 5, 95% of the control and 99% the solvent control larvae had hatched. Therefore, study day 5 was defined as post hatch day 0 (= PHD 0).
The following toxicological endpoints were determined: hatching success, fry growth (assessed via length and fresh weight measurements on PHD 30), morphological and behavioral effects, post-hatch survival and overall survival.
Concentrations of the test item and controls were determined during exposure on day 0, day 7, day 14, day 21 and day 28 for all tested concentration levels and for three constituents C8, C8, C10-BTE, C8, C10, C10 BTE and C10, C10, C10-BTE via LC MS/MS.
1, 2, 4-Benzenetricarboxylic acid, mixed decyl and octyl triesters (CAS-No. 90218-76-1) caused no significant effects on Zebrafish in an early life stage test, 30 days post hatch when tested with nominal concentrations of 7.00, 14.0 and 28.0 µg/L.
For all parameters measured, the nominal NOEC was determined to be ≥ 28.0 µg/L. Corresponding to the arithmetric mean measured concentration NOEC of ≥ 13.9 µg/L of the dispersed fraction of the test item.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.