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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-01-28 to 2019-01-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Principles of method if other than guideline:
Because significant immobility was observed at the two highest examined concentration levels during the Preliminary Range-Finding Test, five test concentrations in a geometric series (factor 3.2) plus a control were used in the Main Test under semi-static conditions. The used factor exceeded the one recommended in the OECD guideline (2.2) due to the observed toxicity in the Preliminary Range-Finding Test.
GLP compliance:
yes
Specific details on test material used for the study:
Name: 2-tert-butyl-1,4-dimethoxybenzene
Public name: Vetimoss
EC number: 244-216-5
CAS number: 21112-37-8
Batch/Lot Number: A180118C
Description:
Clear, pale yellow liquid
Expiry Date: 06 June 2019
Purity: 99.78%
Storage Conditions: Room temperature (15-25°C, ≤70 RH%), under inert gas, protected from humidity (tight closed container).
Analytical monitoring:
yes
Remarks:
HPLC
Details on sampling:
Analytical measurements were performed from the Control and at the applied test concentration levels at the beginning and at the end of each renewal period. Two samples were taken for analytical measurements at 0, 24, 48 hours into 16-mL screw cap glass vials. One set was analysed (Set 1, ~10 mL) and one as a back-up (Set 2, ~10 mL), the weight of the samples was recorded.



Vehicle:
no
Details on test solutions:
Formulation: Because the Test Item is not readily soluble, the the test solutions were prepared individually using a saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23 (2000). The saturated Test Item solutions was prepared individually (mg/L Test Item nominal loading rates (WAFs) by directly dispersing/dissolving the needed amount of Test Item into the test medium (ISO medium) before the start of each renewal period. These solutions were mixed continuously for approximately 1 hour at room temperature. The non-dissolved test materials were removed by filtration through a fine (0.22 µm) filter to give the 100% saturated solutions without an additional settling time. This procedure was considered to be appropriate following an evaluation of the best practical method for this Test Item. These solutions were distributed into the appropriate test flasks at the start of each renewal period (treatment).

Reference Control: For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Untreated Control: The dilution water (ISO-medium) was used without of addition of the Test Item.

Dilution Water: Reconstituted water (ISO medium, according to OECD 202) was used as dilution water for both the range finding and definitive tests. The same composition of reconstituted water was used for the tests and for breeding the test animals.

Dose rates: Because significant immobility was observed at the two highest examined concentration levels during the Preliminary Range-Finding Test, five test concentrations in a geometric series (factor 3.2) plus a control were used in the Main Test under semi-static conditions. The used factor exceeded the one recommended in the OECD guideline (2.2) due to the observed toxicity in the Preliminary Range-Finding Test. The concentrations in the definitive test were based on the results of the preliminary range-finding test and were: 1.0, 3.1, 9.8, 31.3, 100 mg/L nominal loading rates (WAFs).
Test organisms (species):
Daphnia magna
Details on test organisms:
Species and strain: Daphnia magna (Straus)
Source: Szent István University, Department of Aquaculture 2100 Gödöllő, Páter Károly u. 1., Hungary
Breeding: The Daphnia are bred in Ecotoxicological Laboratory of Citoxlab Hungary Ltd.
Justification of species: Daphnia magna is the standard species of the acute immobilisation test.
Previous generation: Daphnia of the previous generation should be reared with conditions similarly to the test conditions as possible. Test daphnia from stage with high reproduction activity (normally 2-4 weeks old) are preferred.
Number of animals: There will be 20 animals at each group (concentration and untreated control) divided into 4 parallel vessels (5 animals/vessel)
Age of animals: Less than 24 hours old at the beginning of the test. The first breeding of the previous generation should not be used.
Acclimatization: To avoid the necessity of adaptation prior to the test, the water used in the test will be similar to the culture conditions (water temperature, background colour etc.).
Holding water: Same water is used as test water.
Food and Feeding: The Daphnia are fed with concentrated algal suspension of Pseudokirchneriella subcapitata during the holding.
Test type:
semi-static
Water media type:
freshwater
Total exposure duration:
48 h
Hardness:
249 mg CaCO3/L
Test temperature:
20.6 – 21.0°C
pH:
7.16 – 7.65
Dissolved oxygen:
7.1– 8.7 mg/L
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0, 1.0, 3.1, 9.8, 31.3, 100 mg/L
Geometric mean measured concentration: 0.29, 0.78, 1.49, 1.50, 4.47 mg/L.
Details on test conditions:
TEST CONDITIONS
Temperature: The water temperature was measured at the start and at the end of each renewal period in each test vessel. The test temperature was in the range of 20.6 – 21.0°C measured in the test vessels.The additionally measured temperature in the climate chamber was between 20.2 – 21.2°C.
Oxygen concentration: The dissolved oxygen concentration was measured in each test vessel at the start and at the end of each renewal period and was in the range of 7.1– 8.7 mg/L.
pH: The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test. The pH was measured at the start and at the end of each renewal period in each test vessel and was in the range of 7.16 – 7.65.
Light: The test was carried out in 16-hour light and 8-hour dark cycle.
Hardness: The reconstituted water (ISO medium) had a total hardness of 249 mg/L (as CaCO3).

PERFORMANCE OF THE TEST
The test duration was 48 hours. Twenty animals, divided into four groups (glass beaker) of five animals each (~8 mL test solution/animal) were used. Taking into account that the stability of the Test Item was not available, the test was performed under semi-static conditions. The frequency of the water renewal periods was 24 hours. The animals were not fed during the test. The choice of the test concentration was done on the basis of the results of a preliminary range-finding test.

OBSERVATIONS
The immobility or mortality of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile. The number of immobilised animals and the percentage of immobility were determined at 24 and 48 hours. The water temperature; the oxygen concentrations and pH of the controls and the test solutions were measured at the beginning and at the end of each renewal period.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
2.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
> 4.47 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
1.49 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
1.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
VALIDITY
There was no immobilized animal in the Control group and the dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

CONCENTRATIONS OF THE TEST ITEM
Test concentrations were analytically determined at the start and at the end of each renewal periods. The LOQ of the method vas 0.21 mg/L and the LOD was calculated as 0.017 mg/L.

The nominal concentrations of Test Item used in the main experiment were: 1.0, 3.1, 9.8, 31.3, 100 mg/L nominal loading rates (WAFs). Test concentrations were analytically determined at the start and at the end of each renewal period. The corresponding measured geometric mean Test Item concentrations were: 0.29, 0.90, 1.49, 1.68, 4.47 mg/L. There were no Test Item detected in the Control samples.

The biological results are based on the measured geometric mean concentrations.

IMMOBILISATION
There was significant effect (20% immobility) at the concentration level of 1.50 mg/L but this was not statistically significantly different, however given the magnitude (20%) and the fact that it was higher than the acceptable Control level immobilization of 10%, therefore this was considered to be the LOELR in this study. The 10% Immobility observed at the concentration levels of 0.78 and 1.49 mg/L were considered to be at the Control level, so the concentration of 1.49 mg/L was set as the NOELR in this study.
Results with reference substance (positive control):
Reference Control: For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study with reference item Potassium dichromate (batch no.: A0345704) is: 23-24 January 2019. The 24h EC50: 0.71 mg/L, (95 % confidence limits: 0.66 – 0.75 mg/L).
Reported statistics and error estimates:
In case of sufficient effects, the ELR50 values will be calculated preferably by Probit analysis with 95% confidence Limits using TOXSTAT or SPSS PC+ software. Other models for ELR50 calculation may be used if appropriate. If a Limit test is performed, no statistical analysis will be necessary. Statistical analysis on immobilization may be assessed by analysis of variance (ANOVA) and Dunnett’s Test (α = 0.05) by TOXSTAT or SPSS PC+ software if necessary. For the determination of the LOELR and NOELR, the immobilization at the test concentrations may be tested on significant differences to the control values by Dunnett’s Test if necessary.

Result of the Preliminary Range-Finding Test

Nominal concentrations

[% saturated solution]

Untreated
control

0.1

1

10

100

Number of
treated / immobilised animals

10 / 0

10 / 0

10 / 0

10 / 6

10 / 10

Exposure concentrations

Nominal concentrations

(mg/L nominal loading rates (WAFs)

Measured concentrations(mg/L)

Measured geometric mean

(mg/L)

1strenewal period

2ndrenewal period

start

end

start

end

Control

n.d.

n.d.

n.d.

n.d.

---

1.0

0.82

0.11¥

0.74

0.11¥

0.29

3.1

2.49

0.25

2.42

0.24

0.78

9.8

4.90

0.46

4.69

0.47

1.49

31.3

6.08

0.60

3.56

0.39

1.50

100

10.33

2.20

8.84

1.99

4.47

n.d.:       not detected

¥:           these values were <LOQ and during the calculation of measured geometric mean concentration, the half of the LOQ was used for calculation of the geometric mean concentration over the exposure period of 72 hours.

Number and percentage of immobilised animals

Concentration

Number of treated animals

Immobilised animals

Nominal

Measured¥

24 hours

48 hours

(mg/L nominal loading rates (WAFs)

(mg/L)

number

%

number

%

Control

--

20

0

0

0

0

1.0

0.29

20

0

0

0

0

3.1

0.78

20

0

0

2

10

9.8

1.49

20

0

0

2

10

31.3

1.50

20

0

0

4

20

100

4.47

20

8*

40

16

80*

* : statistically significantly different compared to the control values (Dunnett’s Test;a= 0.05).

¥ : measured geometric mean concentration.

CONCLUSION

Summary of endpoints

Parameter
(48 hours)

ELR50
(measured)

ELR100
(measured)

NOELR
(measured)

LOELR
(measured)

 

2.70 mg/L

>4.47 mg/L

1.49 mg/L

1.50 mg/L

95 % conf. limits

2.38 – 3.06 mg/L

---

---

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Validity criteria fulfilled:
yes
Conclusions:
The 48-hour daphnia LC50 for Vetimoss is 2.70 mg/l (mean measured concentration).
Executive summary:

In a 48-hour study, the acute toxicity of Vetimoss to daphnia was determined according to OECD 202 guidelines. The 48-hour daphnia LC50 for Vetimoss is 2.70 mg/l (mean measured concentration).

Description of key information

In a 48-hour study, the acute toxicity of Vetimoss to daphnia was determined according to OECD 202 guidelines. The 48-hour daphnia LC50 for Vetimoss is 2.70 mg/l (mean measured concentration).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
2.7 mg/L

Additional information