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Ecotoxicological information

Long-term toxicity to fish

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Administrative data

Endpoint:
fish short-term toxicity test on embryo and sac-fry stages
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP stduy according to protocol.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Chemical name: L-glutamic acid diacetic acid tetrasodium salt
Batch Number: CFC-9131
- Composition of test material, percentage of components:
- Substance (incl impurities) 91.8% w/w
- Water 5.5% w/w
Water solubility: Approx. 1000 g/L
Appearance: Off white powder
pH: 10-12 (1 % solution)
Log Pow: < 0
Expiry date: January 13, 2012

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples of test media including control were taken in 4 sampling intervals from alternating test replicates on days -1, 0 (test start), 5 (test middle) and 9 (test end). Stock solutions were sampled and analysed from one application interval (0 and 5 days old).

All concentration levels and the control were diluted with mobile phase prior to analysis.
All samples were stored at 6 ± 2 °C until start of analysis, if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
Stock solutions were prepared every 3 to 6 days with dilution water. With regard to the water solubility of the test item no organic solvents were used. Syringes were filled in 1-day to 3-day intervals.
5 concentrations were tested in a series with a dilution factor of 2.2. The highest test concentration was tested with pH-adjustment to a range of 7.0 to 8.0 (with 0.1 M hydrochloric acid). Additionally the highest concentration was tested unadjusted (500 mg/L pH 10).
The stock solutions were treated with ultrasound for 60 - 90 minutes at 40°C.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Origin: Niedersächsischer Landesbetrieb für Wasserwirtschaft, Küsten- und Naturschutz, D-31135 Hildesheim, An der Scharlake 39. All fish eggs used in the test were gained at DR.U.NOACK-LABORATORIEN from a single brood stock.

Holding / Brood conditions:
- Temperature: 25 ± 2 °C
- Dissolved oxygen concentration > 60 % of air saturation value
- pH-value: 6 – 8
- Photoperiod: 16 h daily
- Diffuse light (0.1 - 10 µmol photons • m-2 • s-1on water surface)

Food: Artemia salina nauplii, 24 - 48 hours old, ad libitum; dry food sera vipan SERA GMBH, ad libitum.
- No disease treatments were administered.

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. At the end of dawning (3 h after introduction) the glass dishes were gently removed. About 300 eggs were taken and immediately distributed to the test solutions and dilution water (for control).

Fertilization check: After 1 h 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. Fertilisation in the test replicates was 100 %. Afterwards eggs were distributed to the test replicates.

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

Study design

Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
9 d

Test conditions

Hardness:
44.5 mg CaCO3/L
Test temperature:
24.6 - 25.3 °C
pH:
7.34 - 8.38
For 500 mg/L without pH adjustment the pH ranged from 9.64 to 10.09.
Dissolved oxygen:
100% saturation
Nominal and measured concentrations:
Nominal: 21.3, 47.0, 103, 227, 500 (pH 7-8) and 500 (pH 10) mg/L
Measured: 23.3, 49.4, 101, 249, 573 (pH 7-8) and 521 (pH 10) mg/L

Measured concentrations were close to nominal, therefore nominal concentrations were used for determination of the effects.





Details on test conditions:
Test duration: 5 days post hatch (exposure time: 9 days).
Replicates, number of eggs: Three replicates per test and control group with 10 eggs each.
Test vessels: Crystallisation dishes (inner diameter 13.5 cm, water height about 5 cm) were used. The volume of the test media in the dishes was about 700 mL.
Aeration: No aeration was provided.
Equilibration period: The flow through of the test solution and dilution water was started 3 days prior to exposure.
Dilution water: Same as for holding

Test design: A flow-through exposure design was carried out. Membrane piston pumps provided the water flow-through in two consecutively subsequent orders. The first order of membrane piston pumps splitted the dilution water in 6 separate streams. Precision syringe pumps were used for the introduction of stock solutions with different concentrations. The stock solutions and the dilution water were mixed in a mixing chamber by magnetic stirring. This test solution was delivered into a glass aquaria (15 x 15 x 6.5 cm, vol. approx. 1.5 L). A second order of membrane piston pumps provided the test solutions from these aquaria into crystallisation dishes (vol. approx. 0.7 L) where the eggs were exposed. The accuracy of the water flow-through was checked prior to the test start. Water exchange in the delivery glass aquaria was 20 times per day and in the crystallisation dishes 10 times per day, respectively. An equilibration period of 3 days was carried out prior to exposure.

The mean flow rate in the delivery glass aquaria in all test concentrations and the control during the exposure phase was 1.28 ± 0.026 L/h and ranged from 1.17 to 1.32 L/h. In the crystallisation dishes the mean flow rates during the exposure phase were 0.298 ± 0.014 L/h and ranged from 0.263 to 0.315 L/h.

Dilution water:
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

EFFECT PARAMETERS MEASURED:

The number of hatched eggs were determined daily until 5 days after post-hatch.
Post hatch periodL Per definition the post hatch period begun when at least 80 % of all fertilized and living embryos in the control group have hatched
(day 4 of the study).

Mortality: Criteria for mortality vary according to life stage:
- For eggs: Mortality as discerned by a distinct change in coloration or a marked loss of translucency and change in coloration, caused by coagulation and/or precipitation of protein, leading to a white opaque appearance, were checked daily. Dead eggs were discarded.
- For embryos: Absence of body movement and/or heart-beat, change in coloration. Dead embryos were discarded.
- For larvae: Immobility and/or absence of respiratory movement and/or absence of heart-beat (as far as visible) and/or lack of reaction to mechanical stimulus.

Further effects: Abnormal appearance and behaviour were also recorded. The number of larvae or fish showing abnormality of body form and/or pigmentation and the stage of yolk-sac absorption were recorded. Abnormal animals were only removed from the test vessels on death. Abnormalities, e.g. hyperventilation, uncoordinated swimming, swim-up behaviour, atypical quiescence, and atypical feeding behaviour were also be recorded by visually inspecting each replicate.

Length: At the end of exposure (post-hatch day 5) the total length of all survivors were measured to the nearest 0.001 mm.

Body weight: A pooled fish dry weight of each test replicate was determined from all survivors at the end of exposure (post-hatch day 5). Dry biomass weight was measured to the nearest 0.001 mg.
Reference substance (positive control):
not required

Results and discussion

Effect concentrationsopen allclose all
Duration:
9 d
Dose descriptor:
NOEC
Effect conc.:
103 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
number hatched
Duration:
9 d
Dose descriptor:
NOEC
Effect conc.:
94.55 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
5.5% w/w water
Basis for effect:
number hatched
Duration:
9 d
Dose descriptor:
LOEC
Effect conc.:
227 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
number hatched
Duration:
9 d
Dose descriptor:
NOEC
Effect conc.:
>= 500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
length
Duration:
9 d
Dose descriptor:
LOEC
Effect conc.:
> 500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
length
Duration:
9 d
Dose descriptor:
NOEC
Effect conc.:
>= 500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
weight
Duration:
9 d
Dose descriptor:
LOEC
Effect conc.:
> 500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
weight
Duration:
9 d
Dose descriptor:
NOEC
Effect conc.:
227 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: survival (post hatch and overall)
Duration:
9 d
Dose descriptor:
LOEC
Effect conc.:
500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: survival (post hatch and overall)
Duration:
9 d
Dose descriptor:
LC10
Effect conc.:
153 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% C.I. 103-227 mg/L
Duration:
9 d
Dose descriptor:
EC10
Effect conc.:
125 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: inhibition of hatch 0-4 days
Remarks on result:
other: 95% C.I. 103-227 mg/L
Duration:
9 d
Dose descriptor:
EC10
Effect conc.:
114.8 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
5.5% water
Basis for effect:
other: inhibition of hatch 0-4 days
Details on results:
The egg fertilization rate, determined on study day 0 (test start) was > 95 %. The fertilization rate of all test replicates was 100 %.

Egg hatch began on study day 2 in the test item concentrations of 227, 500 mg/L and 500 mg/L (pH 10). Egg hatch in the control group and all other concentrations began on study day 3. Egg hatch of all test replicates continued until study day 5. Study day 4 was determined to be post hatch day 0 (PHD 0) with a control hatching rate of 100 %.

Swim-up was observed for a 5-day period on study days 5 to 9. Newly hatched fry began to swim up on study day 5 (PHD 1). On study day 7 (PHD 3) all surviving larvae had swum up except in the concentration of 500 mg/L, where one larvae in replicate 2 and two larvae of replicate 3 were laying on its side and did not swim up.

The post hatch success in all control replicates met the guideline criteria. The fry survival (post hatch success) at the end of the study was 100 % in the control group, in the test concentrations of 21.3 – 227 – 500 mg/L. In the highest test concentration of 500 mg/L (pH 10) the post hatch success was 0 % at the end of the study.

Overall survival at the end of the study was 100 % in the control group, 93 % in the test concentration of 21.3 mg/L and 103 mg/L, 90 % in the test concentration of 47.0 mg/L, 73 % in the test concentration of 227 mg/L, 70 % in the highest test item concentration 500 mg/L and 0 % in the highest test item concentration of 500 mg/L (pH 10).

No biologically significant morphological and behavioural effects were observed in any tested replicate.
Reported statistics and error estimates:
One way analysis of variance (ANOVA) were used for NOEC/LOEC calculations. A normality test and an equal variance test were done first. For the parameters hatch, growth (length and dry weight), post hatch survival and overall fry survival (mortality), the following statistical tests were conducted:
Hatching data of study days 2 (PHD -2), 3 (PHD -1), 4 (PHD 0) and 5 (PHD 1) were analysed with ANOVA and with a t-test (concentration 227 mg/L and control on days 4 and 5).
Dry weight data, length data, post hatch survival and overall survival (mortality) data of study day 9 (post hatch day 5) were analysed with ANOVA.
Normality tests for hatch of post hatch day -2, post hatch day 0 and post hatch success failed. No transformations were carried out with these data because the data sets were not estimated to follow a normal distribution.
These statistical analyses were conducted with conclusions of statistical significance based on a 95 % confidence level. The α-value was 0.05.

The EC-values for inhibition of hatch (study day 4) and LC-values were calculated with sigmoidal dose response (variable slope) regression.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
GLDA Na4 caused significant effects on the short-term toxicity with embryo and sac-fry stages of zebrafish under flow-through conditions at the dosage levels of 227 - 500 mg/L (nominal test item concentration). Based on the parameters hatch, growth (expressed as length and dry weight), post hatch and overall survival (mortality) the overall NOEC (post hatch day 0 – 5) was 103 mg/L (nominal test item concentration). The overall LOEC was 227 mg/L (nominal test item concentration). The LC10 and the corresponding 95 % confidence interval on post hatch day 5 is 153 (103 227) mg/L (nominal test item concentration). The LC50 and the corresponding 95 % confidence interval on post hatch day 5 is > 500 mg/L (nominal test item concentration). The EC10 and the corresponding 95 % confidence interval on study day 4 (PHD 0) is 125 (103 227) mg/L (nominal test item concentration). The EC50 and the corresponding 95 % confidence interval on study day 4 (PHD 0) is > 500 mg/L (nominal test item concentration). GLDA Na4, applied at a concentration of 500 mg/L, causes an increase of the pH-value, and the toxicity is predominantly based thereon. Only minor effects were found at the highest test concentration of 500 mg/L with an adjusted pH value (in a range of 7 to 8).
Executive summary:

The effects of the test item GLDA Na4 (batch CFC-9131) to the embryo and sac-fry stages of fish (Zebrafish /Danio rerio) were determined according to OECD Guideline 212 from 2010-11-09 to 2010-11-25 with an definitive exposure phase from 2010-11-12 to 2010-11-21at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany.

A flow-through exposure design was performed with the nominal concentrations of 21.3 – 47.0 – 103 – 227 – 500 mg/L (factor 2.2). The highest test concentration was tested with pH-adjustment to a range of 7.0 to 8.0 (with 0.1 M hydrochloric acid). Additionally the highest test concentration was tested unadjusted (500 mg/L (pH 10)).

The test was started by placing fertilized eggs in the test vessels and lasted 9 days (5 days post-hatch). 30 eggs of Danio rerio were exposed to the test concentrations and the control (3 replicates with 10 eggs each).

 

On day four 100 % of the control larvae had hatched. Therefore, study day 4 had been defined as post hatch day 0 (= PHD 0).

 

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

 

The concentration of the test itemand control were analytically verified via HPLC-DAD analysis in 4 sampling intervalsfrom alternating test replicates on days -1, 0 (test start), 5 (test middle) and 9. The stock solutions were sampled and analysed from one application interval.

 

The measured concentrations of GLDA Na4 in the test replicates were in the range of 96 – 119 % of the nominal concentrations throughout exposure. Therefore the acute toxicity of the test itemGLDA Na4 is based on nominal test item concentrations.

 

For the evaluation of the data (determination of the effect level) the test group with 500 mg/L and pH-adjustment to a range of 7 to 8 was included. It is assumed that conditions with a pH-value of 10 which were measured at 500 mg/L without pH-adjustment, will rarely occur in the environment.

The toxicity of GLDA Na4 is predominantly based on the increase of the pH-value. The test item caused 100 % mortality at the test concentration 500 mg/L without any adjustment of the pH-value.