2,4-dimethyl-4-phenyltetrahydrofuran

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 August to 22 September 2014

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:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

date of Inspection: 31 July 2013 / date of Signature: 20/01/10

Analytical monitoring:

yes

Details on sampling:

At the start of the test, ca 200 mg of test substance was dissolved in a final volume of 2000 mL of Elendt M4 medium to give the 100 mg/L test concentration. Aliquots (100, 180, 320 and 560 mL) of the 100 mg/L test concentration were each separately diluted in a final volume of 1000 mL of Elendt M4 medium to give the remainder of the test concentrations of 10, 18, 32 and 56 mg/L, respectively.
Concentrations of test substance in Elendt M4 medium were determined by gas chromatography with mass spectrometric detection (GC-MS) following salination and partitioning into hexane.
At the start of the test (0 hours), 10 mL samples of freshly prepared test media were taken from the control and each test media preparation flask for chemical analysis. At 48 hours, 10 mL samples were also taken for chemical analysis from the pooled expired test media at each test concentration including the control.
In each case duplicate samples were taken, one for chemical analysis and one as a ‘back-up’ should further analysis be required.

Test organisms (species):

Daphnia magna

Details on test organisms:

Juvenile Daphnia magna (Straus), less than 24 hours old, were taken from healthy parental laboratory cultures, held and maintained at the test facility. The culture was originally obtained from Smithers Viscient, Shawbury, UK.
The cultures were originally obtained from Smithers Viscient, Shawbury, UK. The Daphnia magna are cultured in 1 litre glass beakers containing 800 mL of Elendt M4 medium. Each vessel and its contents are referred to as a 'culture'. New cultures are initiated with juvenile Daphnia magna (less than 24 hours old), at a density of approximately 15 daphnids per litre. The cultures are fed daily with a concentrated suspension of Chlorella vulgaris prepared in accordance with standard operating procedures.
The water in each culture was renewed or partially renewed at least twice a week. Juveniles were removed when present in cultures using a sieve. Cultures were maintained up to a maximum of 4 to 5 weeks. Juveniles for use in acute toxicity tests were collected from the second brood onwards. Approximately 24 hours before a test was set up, juveniles present in the cultures were removed and discarded. Over the next 24 hours, juveniles for use in the test were removed from the culture using a wide bore pipette and transferred to fresh culture medium. The juveniles were then left for at least 1 hour before selecting actively swimming individuals for use. All juveniles used to initiate a test were less than 24 hours old.
All cultures, prior to and during toxicity tests, are maintained under fluorescent lighting on a 16-hour light: 8-hour dark photoperiod.
Regular tests are conducted using a reference toxicant to ensure that Daphnia magna cultures are of the highest quality and sensitivity.

Test type:

static

Water media type:

other: Elendt M4 medium

Total exposure duration:

48 h

Test temperature:

19.3-19.6 °C

pH:

7.84-8.10

Dissolved oxygen:

8-9.54 mg/L

Nominal and measured concentrations:

Nominal concentration: 10, 18, 32, 56 and 100 mg/L
Measured concentration: 9.2, 17, 28, 48 and 66 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass jars
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 60 mL
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light: 8-hour dark photoperiod

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): mobility and dissolved oxygen concentration

RANGE-FINDING STUDY
- Test concentrations: 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: 48-hour EC50 > 10 and <100 mg/L.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

24 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Range-finding Test

The results of the range-finding test are summarised below:

Table 6.1.3/1: Immobility of Daphnia magna

Nominal concentration (mg/L)	Cumulative immobilised Daphnia magna*
	24 hours		48 hours		% Immobility
	R1	R2	R1	R2
Control	0	0	0	0	0
0.10	0	0	0	0	0
1.0	0	0	0	0	0
10	0	0	0	0	0
100	5	5	5	5	5

* Initial population of Daphnia magna: five per replicate

R Replicate

 

Table 6.1.3/2: Concentration of test substance

Nominal concentration (mg/L)	Measured Concentration (mg/L)
	0 Hours (New)	48 Hours (Old)
Control	-	-
0.10	0.104 (104)	0.0869 (87)
1.0	0.904 (90)	0.822 (82)
10	38.8 (388)	10.8 (108)
100	79.3 (79)	114 (114)

Values in brackets = % nominal concentration

 

Analysis of the test concentrations at 0 and 48 hours showed measured concentrations to range from 79% to 114% of nominal with the exception of the 10 mg/L test concentration at 0 hours which showed a measured concentration of 38.8 mg/L (388% of nominal). This was considered to be due to an analytical error given the result from the corresponding 48-hour sample which showed a measured concentration of 108% of nominal. It was therefore considered that the test substance was stable in the test system of a 48-hour period.

The results of the range-finding test suggested that the 48-hour EC₅₀ value would be between 10 and 100 mg/L, based on nominal test substance concentrations.

 

Definitive Test

Chemical Analysis

The results of the chemical analysis are presented in the table below.

Table 6.1.3/3: Measured concentrations of test substance in test media during the definitive test with Daphnia magna

Measured Concentration (mg/L)					Geometric mean measured concentration (mg/L)
Nominal concentration (mg/L)	0 Hours (New media)	% of nominal concentration	48 Hours (Old media)	% of nominal concentration
Control	-	-	-	-	-
10	11.9	119	7.12	71	9.2
18	21.5	119	13.2	73	17
32	31.0	97	24.4	76	28
56	58.7	105	39.7	71	48
100	87.4	87	49.1	49	66

“-“Test substance not detected above the limit of quantification (0.002 mg/L)

Analysis of the freshly prepared test media at 0 hours showed measured concentrations to range from 87% to 119% of nominal. Analysis of the old media at 48 hours showed measured concentrations to range from 71% to 76% of nominal with the exception of the 100 mg/L test concentration which showed a measured concentration of 49% of nominal. It was unclear as to why this result was considerably lower than the remaining concentrations. However, as 100% immobilisation was also observed at 56 mg/L it was considered that the results for the 100 mg/L would not affect the calculation of the 48-hour EC₅₀ value and hence the integrity of the test.

Given that a slight decline in measured concentration was observed over the 48-hour test period, it was considered justifiable to base the results on geometric mean measured test concentrations. These were calculated to be 9.2, 17, 28, 48 and 66 mg/L.

 

Water Quality

Water quality determinations for pH, temperature and dissolved oxygen (% ASV and mg/L) during the definitive test are presented in table below

Table 6.1.3/4: percentage immobility

Parameter	Geometric mean Measured concentration (mg/L)	0 hour (new)	48 hours (old)
*Temperature Range (°C)	20.1 – 20.9
Temperature (°C)	Control	19.3	19.6
	9.2	19.3	19.6
	17	19.4	19.6
	28	19.5	19.5
	48	19.5	19.5
	66	19.5	19.5
pH	Control	8.09	7.90
	9.2	8.09	7.85
	17	8.09	7.84
	28	8.09	7.86
	48	8.09	7.85
	66	8.10	7.87
Dissolved oxygen (% ASV and mg/L)	Control	101 (9.39)	90 (8.29)
	9.2	101 (9.43)	87 (8.03)
	17	101 (9.47)	87 (8.12)
	28	101 (9.43)	87 (8.04)
	48	102 (9.52)	87 (8.04)
	66	102 (9.54)	86 (8.00)

* Continuous temperature measurement of the test area by use of a digital maximum / minimum thermometer

ASV: Air saturation value (mg/L value presented in brackets)

 

Test Media Descriptions

The test preparations were observed to be colourless solutions throughout the duration of the test.

 

Toxicity to Daphnia magna

The numbers of immobilised Daphnia magna at 24 and 48 hours, following exposure are presented table below. The percentage immobility data is summarised below.

Table 6.1.3/5: Immobilisation of Daphnia magna after 24 hours exposure during the Definitive test

Geometric mean measured concentration (mg/L)	Number of Daphnia magna exposed	Mobile Daphnia magna		Immobile Daphnia magna
		Submerged	Surface	Submerged	Surface
Control	20	20	-	-	-
9.2	20	20	-	-	-
17	20	20	-	-	-
28	20	20*	-	-	-
48	20	1	-	19	-
66	20	-	-	20	-

- Value not applicable

* Daphnids observed at the bottom of the vessel

 

Table 6.1.3/6: Immobilisation of Daphnia magna after 48 hours exposure during the Definitive test

 

Geometric mean measured concentration (mg/L)	Number of Daphnia magna exposed	Mobile Daphnia magna		Immobile Daphnia magna
		Submerged	Surface	Submerged	Surface
Control	20	20	-	-	-
9.2	20	20	-	-	-
17	20	20	-	-	-
28	20	5	-	15	-
48	20	-	-	20	-
66	20	-	-	20	-

- Value not applicable

 

Table 6.1.3/7: percentage immobility

Geometric mean measured concentration (mg/L)	Number of Daphnia magna exposed	% Immobility at 24 hours	% Immobility at 48 hours
Control	20	0	0
9.2	20	0	0
17	20	0	0
28	20	0	75
48	20	95	100
66	20	100	100

 

Toxicity values and the corresponding NOEC and LOEC values are presented in the following table. Values are presented in terms of geometric mean measured concentrations.

Table 6.1.3/8: geometric mean measured concentrations

	Geometric mean measured concentration (mg/L)
	24-hour	48-hour
EC50	37 (36 – 39)	24 (22 – 27)
LOEC	28	28
NOEC	17	17

Values in brackets are 95% confidence limits

 

After 48 hours, the concentration at which no significant (≤10%) dose related immobility occurred was 17 mg/L. The NOEC was therefore 17 mg/L. After 48 hours 100% immobility was recorded at a concentration of 48 mg/L.

The 48-hour EC₅₀ value was determined to be 24 mg/L.

 

Validity Criteria

The validity criteria for control immobility (≤10%) and dissolved oxygen (>3mg/L) were both satisfied. The test is therefore considered valid.Validity Criteria The validity criteria for control immobility (≤10%) and dissolved oxygen (>3mg/L) were both satisfied. The test is therefore considered valid.

Validity criteria fulfilled:

yes

Conclusions:

The 48-hour acute toxicity of test substance to the freshwater planktonic crustacean, Daphnia magna, was determined in accordance with the requirements of OECD Chemicals Testing Guideline No. 202 Daphnia sp. Acute Immobilisation Test (adopted April 13 2004).
The validity criteria for control immobility (≤10%) and dissolved oxygen (>3 mg/L) were both satisfied.
Based on geometric mean measured concentrations, the 48-hour EC50 value was determined to be 24 mg/L. The corresponding No Observed Effect Concentration (NOEC) was considered to be 17 mg/L.

Executive summary:

The 48-hour acute toxicity of test substance to the freshwater planktonic crustacean, Daphnia magna was conducted in accordance with the requirements of OECD Guideline 202 Daphnia sp. Acute Immobilisation Test.

Based on the results of a range-finding test, for which the key results only have been reported, the definitive test was conducted at nominal concentrations of 10, 18, 32, 56 and 100 mg/L. A control group was also included. The test was conducted using filled and sealed test vessels, to minimise potential losses due to volatility, under static test conditions.

At the start of the test, five juvenile Daphnia magna were added to each test vessel. The Daphnia magna in each test vessel were observed at 24 and 48 hours. The Daphnia magna were not fed during the test.

Analysis of the test media samples was conducted at 0 (fresh media) and 48 hours (old media). Analysis of the freshly prepared test media at 0 hours showed measured concentrations to range from 87% to 119% of nominal. Analysis of the old media at 48 hours showed measured concentrations to range from 71% to 76% of nominal with the exception of the 100 mg/L test concentration which showed a measured concentration of 49% of nominal. It was unclear as to why this result was considerably lower than the remaining concentrations. However, as 100% immobilisation was also observed at 56 mg/L it was considered that the results for the 100 mg/L would not affect the calculation of the 48-hour EC50 value and hence the integrity of the test.

After 48 hours, the concentration at which no significant (≤10%) dose related immobility occurred was 17 mg/L. The NOEC was considered to be 17 mg/L. After 48 hours 100% immobility was recorded at 48 mg/L. In terms of geometric mean measured concentrations the 48-hour EC50 value was determined to be 24 mg/L. The validity criteria for control immobility (≤10%) and dissolved oxygen (>3 mg/L) were both satisfied.

Description of key information

OECD Guideline 202, GLP, key study, validity 1:

48h-EC50 (Daphnia magna) = 24 mg/L based on measured geometric mean concentration

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

24 mg/L

Additional information

To assess the short-term toxicity of the registered substance to aquatic invertebrates, one key experimental study is available.

The 48-hour acute toxicity of test substance to the freshwater planktonic crustacean, Daphnia magna was conducted according to OECD Guideline 202 with GLP statement.

Based on the results of a range-finding test, for which the key results only have been reported, the definitive test was conducted at nominal concentrations of 10, 18, 32, 56 and 100 mg/L. A control group was also included. The test was conducted using filled and sealed test vessels, to minimise potential losses due to volatility, under static test conditions.

 

At the start of the test, five juvenile Daphnia magna were added to each test vessel. The Daphnia magna in each test vessel were observed at 24 and 48 hours. The Daphnia magna were not fed during the test.

 

After 48 hours, the concentration at which no significant (≤10%) dose related immobility occurred was 17 mg/L. The NOEC was considered to be 17 mg/L. After 48 hours 100% immobility was recorded at 48 mg/L. In terms of geometric mean measured concentrations the 48-hour EC50 value was determined to be 24 mg/L. The validity criteria for control immobility (≤10%) and dissolved oxygen (>3 mg/L) were both satisfied.