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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17.9.-8.10.1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study approved by authority in ELINCS notiifcation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OECD guideline No. 202: "Daphnia sp., Acute Immobilisation Test and Reproduction Test", Adopted April 4, 1984, extended with recommendations and supplements which are included in the Draft proposal for an update of DECD Guideline 202, Part II (1991).
Qualifier:
according to guideline
Guideline:
other: EEC Draft guideline XI/681/86: "Prolonged Toxicity Study with Daphnia magna: Effects on Reproduction"; Version 4
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(phenylmethoxy)naphthalene
EC Number:
405-490-3
EC Name:
2-(phenylmethoxy)naphthalene
Cas Number:
613-62-7
Molecular formula:
C17H14O
IUPAC Name:
2-(benzyloxy)naphthalene
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): BNE
- Substance type: organic
- Physical state: solid
- Analytical purity: 99.8 %
- Lot/batch No.: 20979
- Expiration date of the lot/batch: August 31, 1994
- Storage condition of test material: at room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: samples were taken from the filtrate and three concentrations: the lowest, a middle and the highest concentration.
- Sampling method:
- Sample storage conditions before analysis: Samples were transferred to Analytical Chemistry on the day of sampling.
Reserve samples were stored in deep freeze for possible analysis.

Test solutions

Vehicle:
yes

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST SYSTEM
- Species: Daphnia·magna (Crustacea, Cladocera) (Straus, 1820)
- Reason for selection: This system has been selected as an internationally accepted species.
- Validity of batch: Frequent inspection of the cultures with respect to number of young, appearance of young and parental Daphnia
and general feeding behaviour.
- Characteristics: For the test selection of young daphnids with an age of <24 hours.

BREEDING
- Start of each batch: With newborn animals, i.e. less than 3 days old, by placing them individually in 50 ml M4 medium.
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: Two times a week.
- Temperature of medium 18-22°C, constant within ± 1°C
- Feeding Daily, a suspension of fresh water algae (Chlorella pyrenoidosa).
- Medium: M4, as prescribed by Dr. Elendt-Schneider
(Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultra-structural approach to antennal damage in Daphnia magna, Straus).
Composition of medium M4:
- ISO-medium: The following chemicals (analytical grade) are dissolved in freshly prepared ultra-pure water
(mg/l):
CaC12·2H20 293.8
MgS04·7H20 123.3
NaHC03 64.8
KCl 5.8
- Medium M4: Trace elements, macro nutrients and vitamins are added to freshly prepared ISO-medium to reach the following concentrations:
Trace elements (mg/l):
B 0.5
Fe 0.2
Mn 0.1
Li, Rb and Sr 0.05
Mo 0.025
Br 0.0125
Cu and Zn 0.0063
Co and I 0.0025
Se 0.0010
V 0.0003
Na2EDTA.2H2O 2.5
Macro nutrients (mg/l):
Na2SiO3.)H2O 10.0
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18
Vitamins (µg/l):
Thiamine 75.0
B12 1.0
Biotin 0.75

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d

Test conditions

Hardness:
250 mg/l CaCO3
Test temperature:
between 19.5 and 21 °C
pH:
8.2 - 7.5

The deviations between the pH values of the fresh media remained ~ 0.3 units as recorded in the control and the highest test concentration (180 µg/l).
During the first part of the test, pH decreased by 0.1 to 0.4 unit during the periods before renewal of the test solutions (Old media).
During the second part, pH decreased by 0.4 to 0.6 unit during the periods before renewal. This was consistent with the recorded decreases of dissolved oxygen during these periods.
Dissolved oxygen:
5.3 - 9.3 mg/l
Nominal and measured concentrations:
nominal: Test concentrations 7.2, 16.2, 36, 81 and 180 µg/l
Control: Test medium without test substance or other additives
Test medium without test substance but with 0.03 ml/l Tween 80
For comparison of nominal and measured concentrations refer to Any other information on materials and methods incl. tables.
Details on test conditions:
TEST SYSTEM
- Test vessel: Volume: 100 ml (8 x d 4 cm), all-glass covered with a glass plate. Each test vessel contained 50 ml of test solution.
- Renewal of the test media: Three times a week (Monday, Wednesday and Friday)
- No. of organisms per vessel: 10 neonate (< 24h old) daphnids per test concentration,
while the control groups consisted each of 20 daphnids.
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 1


TEST MEDIUM: M4
WATER PARAMETERS
- Source/preparation of dilution water: Tap water purified by reverse osmosis and
subsequently passed over activated carbon and ion-exchange cartridges: Milli-Q water (Millipore Corp., Bedford, Mass., USA)
PREPARATION OF TEST MEOIA
The test media were prepared using fresh stock solutions with a nominal BNE concentration of 100 mg/l in medium M4.
Weighed amounts of BNE were first mixed with Tween 80. Subsequently this mixture was dispersed in medium M4.
The dispersions were stirred for 4B-72 hours and then filtered through a 5 µm filter. Exact aliquots were taken from the filtrate and diluted in
medium M4 for the preparation of the test solutions. The final test solutions contained 0.4, 0.9, 2.0, 4.5 and 10 %of the filtrate.
An additional stock was prepared containing 0.3 ml Tween 8 per l M4-medium.
Exact aliquots of this stock solution were added to the test solutions to provide a final Tween 80 concentration of 0.03 ml/l in all BNE treated
solutions.
All test solutions were clear and there was no indication of precipitation of the test material.


OTHER TEST CONDITIONS
- Photoperiod: 16 hours daily
- Light intensity: 220 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Parental Daphnia
- Immobility and mortality: Three times a week at each renewal, the number of living, immobile and dead parental daphnids were recorded.
Dead daphnids were removed when observed.
Also, the presence of eggs in the brood pouch was recorded three times a week.
F1 generation
- Appearance first brood: When observed.
- Newborn daphnids: Three times a week at each renewal the number of newborn young was counted
and the condition of the young recorded. Thereafter the young were removed.
- Presence of unhatched eggs: Three times a week at each renewal.
- Incidence of immobility: Three times a week at each renewal.




Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
132 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: parental immobility
Remarks on result:
other: 95 % fiducial limits 98 - 227 µg/l
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
180 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: parental immobility
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
81 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: parental immobility
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
136 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95 % fiducial limits 73 - 303 µg/l
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
81 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
36 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
- Immobility including mortality of parent animals: refer to Table 2
During the 21 day exposure period, 70 % of the parental Daphnia exposed to 180 µg/l became immobilized.
No significant (>20%) immobilisation (including mortality) was recorded at any of the other test concentrations (Fisher Exact test: P=0.05).

- No. of offspring produced per day per female: refer to table 4
The mean number of living offspring recorded for the Tween control was substantially lower than that recorded for the blank control.
Further, the standard deviation for the Tween control was greater than 25% of the mean (67%)
owing to very low numbers of living offspring produced by 4 out of 16 surviving parental daphnia.
Comparison of the means of the various test groups with that for the Tween control showed that:
1. The production of living offspring was substantially lower at 81 and 180 µg/l during the exposure period,
whereas the other treated groups produced higher average numbers of offspring from day 14 onwards.
2. The reproductive capacity of the parental daphnia of at 81 µg/l tended to recover during the last week of exposure.
3. At the end of the 21-day test period, only the average reproduction of parents exposed to 180 µg/l was
more than 25% lower than that of the Tween control .
The significance of the reduction of reproductive capacity was confirmed by statistical analysis using the Williams' test (P=0.05).
Finally, comparison of the mean numbers of living offspring recorded showed
no test concentration related effect on reproduction by BNE up to and including 36 µg/l. Hence, although not statistically significant, BNE affected the reproductive capacity of Daphnia magna at 81 µg/l (nominal).

- Time to first brood release: refer to table 3
Test substance related delay of first brood was recorded at BNE concentrations of 81 and 180 µg/l.

- Effect concentrations exceeding solubility of substance in test medium: yes
Reported statistics and error estimates:
Statistical analysis:
The incidences of parental mortality in the different test groups were statistically tested against that recorded in the Tween control
using the Fishers Exact Test.
The values for reproduction of the solvent control were compared with the blank control using the t-test. Further, for each concentration the results
of reproduction were tested for normality and for homogeneity of variance. Then these data were statistically tested using the Williams' Test.
The overall threshold level of effect and the overall NOEC were determined on basis of these statistics. Further, the EC50 for parental immobility and
the EC50 for reproduction were calculated (Finney, D.J., 1971: Probit analysis, Cambridge University Press, Cambridge, U.K., 3rd edition).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
In general, all test conditions remained within the protocolled ranges except pH. The measured pH ranges exceeded 0.3 units, but remained within 0.6 units and was related to the experimental design of the test.
Conclusions:
The EC50 of 2-(phenylmethoxy)naphthalene for parental immobility was in the same range as the EC50 for reproduction.
Both values exceeded the maximum solubility of 2-(phenylmethoxy)naphthalene in water (ca. 70 ~g/l).
Test concentrations above this level could only be prepared using a dispersing agent (Tween 8O). The applied Tween 8O concentration of 0.03 m1/1 in all 2-(phenylmethoxy)naphthalene treated solutions had a significant effect on the reproductive capacity of Daphnia magna. Hence, the presence of Tween 8O probably increased the susceptibility of the organisms to the 2-(phenylmethoxy)naphthalene concentrations resulting in an overestimation of the toxic effect of 2-(phenylmethoxy)naphthalene on the reproductive capacity of Daphnia magna.
Due to its low solubility, BNE itself may not be available to the test organisms to cause a toxic effect on the reproductive capacity in a spill or other discharge type situation.
Executive summary:

2-(phenylmethoxy)naphthalene was investigated for possible effects on survival and reproductive performance in Daphnia magna during 21 days of exposure.

The experiment started with 10 vessels per concentration each containing one neonate (24h old) Daphnia magna in 50 ml test media. The test solutions consisted of 5 dilutions of a filtrate from a dispersion of 100 mg 2-(phenylmethoxy)naphthalene per litre. 2-(phenylmethoxy)naphthalene was dispersed using Tween 80 as a dispersing agent at a final concentration of 0.03 ml/l present in all 2-(phenylmethoxy)naphthalene treated solutions.

The test solutions were renewed three times a week. At the start of the test and at each renewal the condition of the parental daphnia was recorded and during the reproduction phase the number of living offspring, immobile young and number of unhatched eggs were recorded.

Analysis of the samples taken at the start and the end of the test showed that 2-(phenylmethoxy)naphthalene concentrations did not decrease by more than 20%

during the first renewal (72 hours) and were ~ 80 % of the expected concentrations based on measurement of samples taken from the filtrate. However, analysis during the 48 hours after the last renewal showed non quantifiable concentrations on day 21, whereas analysis of samples taken from the fresh media on day 19 showed measured concentrations of approximately 100 % relative to the expected values. No explanation could be given for this high decrease in measured concentration.

The mean number of living offspring recorded for the Tween control was substantially lower than that recorded for the blank control. Further, the standard deviation for the Tween control was greater than 25% of the mean owing to very low numbers of living offspring produced by 4 out of 16 surviving parental daphnia. Only the average reproduction of parents exposed to 180 µg/l was more than 25% lower than that of the Tween control.

The significance of this reduction of reproductive capacity was confirmed by statistical analysis using the Williams' test. Further, no test concentration related effect on reproduction by 2-(phenylmethoxy)naphthalene was recorded up to and including 36 µg/l. However, although not statistically significant, 2-(phenylmethoxy)naphthalene affected the reproductive capacity of Daphnia magna at 81 µg/l (nominal).

The EC50-values for parental mobility and reproduction, the lowest observed effect concentrations (LOEC) and the overall no observed effect concentration (NOEC) based on nominal concentrations are shown below:

 

Parameter

Nominal BNE concentration(µg/l)

21-day EC50 for parental immobility

132

95 % fiducial limits

98 - 227

LOEC for parental immobility

180

NOEC for parental immobility

81

EC50 for reproduction

136

95 % fiducial limits

73 - 303

LOEC for inhibition of reproduction

81

NOEC for inhibition of reproduction

36

Final considerations:

The EC50 of 2-(phenylmethoxy)naphthalene for parental immobility was in the same range as the EC50 for reproduction. Both values exceeded the maximum solubility of 2-(phenylmethoxy)naphthalene in water (ca. 70 µg/l). Test concentrations above this level could only be prepared using a dispersing agent (Tween 80). The applied Tween 80 concentration of 0.03 m1/1 in all 2-(phenylmethoxy)naphthalene treated solutions had a significant effect on the reproductive capacity of Daphnia magna. Hence, the presence of Tween 80 probably increased the susceptibility of the organisms to the 2-(phenylmethoxy)naphthalene concentrations resulting in an overestimation of the toxic effect of 2-(phenylmethoxy)naphthalene on the reproductive capacity of Daphnia magna. Due to its low solubility, 2-(phenylmethoxy)naphthalene itself may not be available to the test organisms to cause a toxic effect on the reproductive capacity in a spill or other discharge type situation.