3,4-xylenol

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation: 2021-10-07
Experimental starting: 2021-11-10
Experimental completion: 2021-11-15

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

99% purity, GC
Batch SHBL3034
Expiry date: 2024-06-01

Analytical monitoring:

yes

Details on sampling:

All concentration levels and the control were analytically verified via GC-MS in the fresh media at the start of exposure and at the renewal of the test solutions (0 and 24 hours) as well as in the 24-hours old media at the renewal and at the end of the exposure (24 and 48 hours).

At the start, samples were taken after preparation of each concentration level and the control and analyzed. At the end of the renewal (24 hours) and the exposure (48 hours), samples of the old media were taken from the test vessels.

Details on test solutions:

Stock solution, media preparation: 5.00 mg test item/L were freshly prepared with dilution water. The dispersion was treated with ultrasound for 10 minutes at room temperature.

Test organisms (species):

Daphnia magna

Details on test organisms:

Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany
Breeder: Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany
Culture: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20°C (+/- 2°C), 16 hours illumination, light intensity of max. 1500 lx
Culture medium: Elendt M4, according to OECD 202, Annex 3 (2004), is used.
Feeding of the culture stocks: The daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus. The algae were cultured at the test facility.

Less than 24 hours old daphnids from a healthy stock were used for the study. To achieve this, juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period were used for the test. No first brood progeny was used for the test.

Acclimatization was not necessary, because the composition of the dilution water is equivalent to the culture medium.

Test type:

semi-static

Water media type:

freshwater

Remarks:

Same composition as the culture medium

Limit test:

no

Total exposure duration:

48 h

Hardness:

new media: 242 mg/L as CaCO3
old media: 257 mg/L as CaCO3

Test temperature:

18-22°C, constant within 1°C

pH:

new media: 7.45-8.01
old media: 7.69-7.88

Dissolved oxygen:

new media: 7.08-8.94 mg/L
old media: 8.24-9.07 mg/L

Conductivity:

new media: 586 µS/cm
old media: 629 µS/cm

Nominal and measured concentrations:

Nominal: Out of the stock solution 5 concentrations were tested in a geometric series with a dilution factor of 4√10: 0.500 – 0.889 – 1.58 – 2.81 – 5.00 mg/L.

Measured (new media): 0.468, 0.707, 1.89, 3.28, 5.25 mg/L
Measured (old media): 0.457, 0.836, 1.62, 2.72, 5.19 mg/L
% of nominal: 80-117%

Details on test conditions:

TEST SYSTEM
- Test vessel: Sealed glass flasks (4.5 (ID) x 9.5 (H) cm) with screw were used and filled up to the top with the test solutions. A test volume of approximately 130 mL was provided in each test vessel.
- Type: closed
- Volume of solution: Approx. 130 mL
- Aeration: None
- Renewal rate of test solution: renewal after 24h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Dilution water: Culture Medium Elendt M4 (according tot OECD 202, Annex 3, 2004)
- Culture medium different from test medium: no
- Intervals of water quality measurement: 24 h

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: /8 hours light/dark cycle
- Light intensity: diffuse light, intensity of max.1500 lx


RANGE-FINDING STUDY
A non-GLP preliminary range finding test was conducted at the test facility under semi-static conditions in a closed system without headspace over a period of 48 hours. A stock solution with a concentration of 50 mg/L was freshly prepared with dilution water and treated with ultrasound at room temperature for 10 minutes. The stock solution was further diluted to the concentration levels 0.500 and 5.00 mg/L with dilution water. All tested concentration levels were visually clear and colorless throughout the exposure period.
The preliminary range finding test was conducted under diffuse light conditions (light intensity of max. 1500 lx, 16/8 hours light/dark cycle).
In the range finding test, two replicates per concentration level and control, each with five daphnids, were tested. 100% mprtality was observed after 24h in the 50 mg/L exposure. Partial mortality (40%) was noted after 24g in the 5 mg/L exposure, and thi % increased to 90% after 48h. No mortality was found in the 0.5 mg/L exposure.

Reference substance (positive control):

yes

Remarks:

A reference test with potassium dichromate was conducted as an acute immobilization test (acc. to AQS P 9/2 and OECD 202) in Elendt M4 medium under static conditions with a test duration of 24 hours

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.36 mg/L

95% CI:

2.82 - 4.95

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

measured values were within 80-104% of nominal values

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

4.91 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

2.69 mg/L

95% CI:

2.69 - 4.89

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Immobilization was determined in all groups after 24 and 48 hours. A daphnid was considered immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel. Observation of other adverse, sub-lethal effects (e.g. discoloration, abnormal behavior etc.) was done, but none occurred during the course of the study.

The measured water quality parameters (i.e. pH-value, dissolved oxygen concentration, total water hardness and water temperature) were within the acceptable limits during the study. During the test period, the temperature in the incubator was 20 - 21 °C.

The concentrations of the test item 3,4-xylenol were analytically verified via GC-MS/MS in the fresh media at the start of the exposure and at the renewal of the test solutions (0 and 24 hours) and in the 24-hours old media at the renewal and at the end of the test (24 and 48 hours) in all concentration levels and the control. The measured concentrations of the test item 3,4-xylenol in the fresh media were in the range of 80 to 120% of the nominal values at the start of the exposure (0 hours) and at the renewal (24 hours). The measured concentrations of the test item in the old media at the renewal and at the end of the test (24 and 48 hours) were in the range of 91 to 104% of the nominal values.
Procedural recovery samples (QC) were prepared on the 10 x LOQ level and treated in parallel to the test item samples. Mean recovery was 107% and confirmed therefore the validity of the analytical method.

Results with reference substance (positive control):

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number BCCC1619, purity 100%, CAS RN 7778-50-9) was determined after 24 hours (2021-10-12 to 2021-10-13), and resulted in a 24h-EC50 of 1.57 mg/L (CL: 1.0 -4.0 mg/L). The valid range is between 0.6 and 2.4 mg/L.

Reported statistics and error estimates:

The EC10-value after 24 hours and the EC10/50-values after 48 hours of exposure were calculated by sigmoidal dose-response regression with the software GraphPad Prism. The EC50-value after 24 hours was estimated empirically from the observed immobilization rates. The respective 95% confidence limits for the EC10-value after 24 hours and for the EC10/50-values after 48 hours were calculated from the standard error and the t distribution wherever possible. In the case that the confidence limits could not be calculated by the software, the respective confidence limits were empirically derived from the observation data. All calculations were carried out from the best-fit values with the software GraphPad Prism.

All data were computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software:
1) GraphPad Prism, GRAPHPAD SOFTWARE, INC.
2) Excel, MICROSOFT CORPORATION

Validity criteria fulfilled:

yes

Remarks:

The dissolved oxygen concentrationis ≥ 3 mg/L in all concentration levels and in the control. The pH is between 6 – 9. The deviation of the final pH-values is less than 1.5.

Conclusions:

An acute immobilization test to Daphnia magna (Straus) carried out to determine the EC10 / 50 / 100-values of 3,4-xylenol after 24 and 48 hours of exposure under semi-static conditions in a closed system without headspace. A reliable 48h-EC50 of of 3.36 mg/L (95%CL 2.82-4.95 mg/L) was determined, and this value can be used for the environmental hazard assessment of 3,4-xylenol.

Executive summary:

In the acute immobilization test with Daphnia magna, the acute toxcicity of the test item 3,4-xylenol,  was determined at the test facility according to OECD 202 (2004). The study was conducted under semi-static conditions in closed test vessels without headspace over a period of 48 hours. Five concentrations of the test item  prepared from a stock solution of 5.00 mg test item/L in a geometric series with a separation factor of ⁴√10 were tested. Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control.

The concentrations of the test item 3,4-xylenol were analytically verified via GC-MS/MS in the fresh media at the start of the exposure and at the renewal of the test solutions in all concentration levels and the control. The measured concentrations of the test item 3,4-xylenol  in the fresh media were in the range of 80 to 120% of the nominal values at the start of the exposure (0 hours) and at the renewal (24 hours). The measured concentrations of the test item in the old media at the renewal and at the end of the test (24 and 48 hours) were in the range of 91 to 104% of the nominal values. The EC_x-values are based on the nominal concentrations of the test item 3,4-xylenol. The validity criteria of the test guideline were fulfilled. A 48h-EC50 of 3.36 mg/L (95%CL 2.82-4.95 mg/L) was determined for 3,4-xylenol.

 

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Originally present in AQUIRE database. All AQUIRE data entries have been subjected to established quality assurance procedures. Data used for the harmonised classification of Index No 604-006-00-X (which includes 3,4-xylenol).

Qualifier:

according to guideline

Guideline:

other: Info not available

GLP compliance:

not specified

Remarks:

ECOTOX database does not provide info on GLP. Original study published in 1979

Analytical monitoring:

yes

Test organisms (species):

other aquatic crustacea: Crangon septemspinosa

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

13.7 mg/L

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Originally present in AQUIRE database. All AQUIRE data entries have been subjected to established quality assurance procedures. Data used for the harmonised classification of Index No 604-006-00-X (which includes 3,4-xylenol).

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

Info not available

Principles of method if other than guideline:

- Principle of test: 50% immobilization concentration at 24 h was determined for Daphnia magna under static conditions

GLP compliance:

not specified

Remarks:

ECOTOX database does not provide info on GLP.

Analytical monitoring:

no

Test organisms (species):

Daphnia magna

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

24 h

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

18.7 mg/L

Basis for effect:

mobility

Description of key information

The data of studies studies with C.septemspinosa and D. magna (Ecetox, 2019) were used for the harmonised classification of Index No 604-006-00-X (which includes 3,4-xylenol). A new study with D.magna (NOACK, 2021) confirmed the harmonized acute/chronic hazard classification.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

3.36 mg/L