Dihexyl ether

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analysis of the test media samples was conducted on fresh media on Days 0, 7, 8, 14 and 20 and on corresponding pooled old media on Days 1, 8, 9, 15 and 21 for all test concentrations. 10 ml samples were taken at each sampling. The samples were frozen over the test duration until analysis at the test end (sample stability confirmed for 21d).

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances) :
A 10 mg/L stock solution was prepared by direct addition of test substance to Elendt M4 medium. The bottle was sealed and stirred in the dark for ca 1 hour. After stirring, the test solution was settled for 1 hour in the dark and then test solution was removed by siphoning. The removed test solution was vacuum filtered using 0.45 μm cellulose nitrate membrane filters to produce the 100 % saturated solution. The 100 % saturated solution was diluted with filtered Elendt M4 medium to prepare the definitive test concentrations.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Waterflea
- Justification for species other than prescribed by test guideline: not applicable
- Source: MicroBioTests Inc., Belgium
- Feeding during test: no


CULTURE
The Daphnia magna were cultured in 1 litre glass beakers containing 800 mL of Elendt M4 medium. New beakers were initiated with juvenile Daphnia magna (less than 24 hours old), at a density of approximately 10 - 15 daphnids per litre. The cultures were fed daily with a concentrated suspension of Chlorella vulgaris.
Juveniles for use in acute toxicity tests were collected from the second brood onwards. Approximately 24 hours before the 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.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

no

Hardness:

> 140 mg CaCO3/l

Test temperature:

18 - 22°C

pH:

7 – 9

Dissolved oxygen:

> 5.8 mg/l

Nominal and measured concentrations:

The time-weighted mean measured concentrations were 0.000303, 0.00106, 0.00260, 0.0115 and 0.0361 mg/L for the 0.5, 1.6, 5.0, 16 and 50 % saturated solutions, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 ml glass screw top glass jars
- Type: closed, no headspace
- Aeration: no
- Renewal rate of test solution (frequency): after 24h
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10 (non-filtered)
- No. of vessels per additional control (replicates): 10 (filtered)

TEST MEDIUM / WATER PARAMETERS
- Standard test medium: yes (Elendt M4 medium)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16h light - 8h dark
- Light intensity: 1000-1500lux

EFFECT PARAMETERS MEASURED:
survival, growth, time to first brood and juvenile production per surviving parental daphnid

RANGE-FINDING STUDY
Based on the results of the Daphnia acute test, the definitive test was conducted at nominal saturated concentrations of 0.5, 1.6, 5.0, 16 and 50 % saturated solutions. A control and filtered control treatment was also included in the test.

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.014 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: (0.00384 – 0.0212, 95% CI)

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.011 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

No significant effects on the adult survival were observed at any test concentration except the when compared to the filtered control treatment. High mortality (40%) was observed in the 0.00106 mg/L treatment at the end of the test. This mortality was not considered to be test substance related, as the mortality in all other test treatments was ≤20%, therefore, indicating this result to be anomalous. This mortality was confirmed to not be statistically significant when compared with the filtered control.

Reported statistics and error estimates:

Formal statistical analysis was conducted on the data using the statistical software package CETIS v1.8.6.8. The test concentration results were compared to the filtered control results. All results were based on time weighted mean measured concentrations.
The adult survival NOEC and LOEC was calculated using the Fishers Exact/Bonferroni-Holm Test. The adult growth NOEC and LOEC was calculated using the Bonferroni Adj t Test. The time to first brood NOEC and LOEC was calculated using Jonckheere-Terpstra Step-Down Test. The juvenile production NOEC and LOEC was calculated using the Wilcoxon/Bonferroni Adj Test. The adult survival, growth and juvenile production EC10, 20 and 50 values were calculated using Linear Interpolation (ICPIN).

Time-Weighted Mean Measured Concentrations (mg/L)

	Adult Survival	Adult Length	Time to First Brood	Reproduction (Juvenile Production)
EC10	> 0.0361	> 0.0361	> 0.0361	0.0136
EC20	> 0.0361	> 0.0361	> 0.0361	0.0249
EC50	> 0.0361	> 0.0361	> 0.0361	> 0.0361
NOEC	0.0361	0.0361	0.0361	0.0115
LOEC	> 0.0361	> 0.0361	> 0.0361	0.0361

Validity criteria fulfilled:

yes

Conclusions:

The EC10 for juvenile production was estimated to be 0.0136 mg/l, based on time-weighted mean measured concentrations.

Executive summary:

The objective of the study was to determine the effects of Dihexyl Ether on Daphnia magna reproduction in accordance with the OECD Chemicals Testing Guideline No. 211 Daphnia magna Reproduction Test (October 2012). The test was conducted at nominal saturated concentrations of 0.5, 1.6, 5.0, 16 and 50 % saturated solutions. A control and filtered control treatment was also included in the test.

In an effort to maintain the test concentrations, the test was conducted in completely filled and sealed test vessels. The test was conducted using a semi-static design with a renewal of the test media at 24-hour intervals. Ten replicate test vessels were prepared for each treatment and control groups. A single juvenile Daphnia magna (<24 hours old) was added to each test vessel.

The Limit of Quantification (LOQ) was 0.001 mg/L for the analytical method. Analysis of the test media samples was conducted on fresh media on Day 0, 7, 8, 14 and 20 and on corresponding pooled old media on Day 1, 8, 9, 15 and 21. The results of the chemical analysis showed that the test substance was not stable over the 24-hour renewal period, as the test concentration declined significantly over each 24-hour renewal period. Due to the significant decline in test concentration, the measured concentrations have been reported as time-weighted mean measured concentrations. The time-weighted mean measured concentrations were 0.000303, 0.00106, 0.00260, 0.0115 and 0.0361 mg/L for the 0.5, 1.6, 5.0, 16 and 50 % saturated solutions, respectively.

The Day 21 EC50, NOEC and LOEC toxicity values in terms of survival, growth, time to first brood and juvenile production per surviving parental Daphnia magna were derived statistically and are based on time weighted mean measured concentrations (mg/L).

Mortality of Daphnia magna

The NOEC value for adult survival was considered to be 0.0361 mg/L based on time weighted mean measured concentrations. The EC10, EC20 and EC50 for adult survival was considered to be greater than 0.0361 mg/L, respectively, based on time-weighted mean measured concentrations.

Growth of Daphnia magna

No significant effects on the growth were observed at any test concentration when compared to the filtered control treatment. The NOEC value for growth was considered to be 0.0361 mg/L based on

time-weighted mean measured concentrations. The EC10, EC20 and EC50 for growth was considered to be greater than 0.0361 mg/L, respectively, based on time-weighted mean measured concentrations.

Time to First Brood

No significant effects on the time to first brood were observed at any test concentration when compared to the filtered control treatments. The NOEC value for time to first brood was considered to be 0.0361 mg/L based on time-weighted mean measured concentrations. The EC10, EC20 and EC50 for time to first brood was considered to be greater than 0.0361 mg/L, respectively, based on time-weighted mean measured concentrations.

Daphnia magna Juvenile Production

Statistically significant effects on juvenile production were observed at the highest test concentration (0.0361 mg/L) when compared to the filtered control treatments. The NOEC value for juvenile production was considered to be 0.0115 mg/L based on time weighted mean measured concentrations. The EC10, EC20 and EC50 for juvenile production was estimated to be 0.0136, 0.0249 and > 0.0361 mg/L, respectively, based on time-weighted mean measured concentrations.

The validity criteria for control mortality was ≤20% and the mean number of live offspring produced per parental Daphnia magna surviving at the end of the test was greater than 60, therefore the test was considered valid.

Description of key information

Based on an OECD 211 Daphnia magna Reproduction Test, the EC10 value for reproduction was determined to be 0.0136 mg/L based on time weighted mean measured concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.014 mg/L

Additional information

A further OECD 211 Daphnia magna Reproduction Test resulted in a NOEC(21d) of 0.020 mg/l, hence supporting the determined EC10 of the key study.

In addition to the experimental values, a reliable QSAR model (Ecological Structure-Activity Relationship (ECOSAR) version 1.11) was used to predict the chronic aquatic toxicity of dihexyl ether to daphnids. The daphnid 21 day ChV was 0.051 mg/L for the neutral organic class. The ChV was divided by√2 to estimate a NOEC equivalent value of 0.036 mg/l, further supporting the experimental observations.