Bis[2-(2-ethoxyethoxy)ethyl] cyclohexane-1,4-dicarboxylate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 March 2011 - 6 May 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

: At the end of the limit test the mean cv for section-by-section specific growth rates in the control cultures was > 35% due to low growth rate during the first 24 h of exposure, but effect was observed in both control and limit group: test is acceptable

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

(2008), amended by EC No. 761/2009

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Neosolue-Aqulio

IUPAC Name:

Neosolue-Aqulio

Constituent 2

Reference substance name:

Bis[2-(2-ethoxyethoxy)ethyl] cyclohexane-1,4-dicarboxylate

EC Number:

618-829-7

Cas Number:

922165-31-9

Molecular formula:

C20H36O8

IUPAC Name:

Bis[2-(2-ethoxyethoxy)ethyl] cyclohexane-1,4-dicarboxylate

Details on test material:

- Name of test material (as cited in study report): Neosolue-Aqulio
- Description/Physical state: Clear colourless to very pale yellow liquid
- Molecular formula: C20H36O8
- Molecular weight: 404.5
- Analytical purity: 99.5% (GC)
- Lot/batch No.: TVI-714
- Expiration date of the lot/batch: 07 October 2012 (Retest date)
- Storage condition of test material: At room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/l and control (0 mg/l) at t=0, t=24 and t=72 h
- Sampling method/volume: 2 ml
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Test solutions

Details on test solutions:

PREPARATION OF TEST SOLUTION:
- method: Test substance was stirred in medium at a nominal concentration of 100 mg/l applying 15-20 minutes of magnetic stirring to accelerate the dissolution. The lower test concentrations for the range-finding test were prepared by subsequent dilutions of the 100 mg/l concentration in test medium. The final test solutions were all clear and colourless.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

After preparation, volumes of 50 ml test solution were added to each replicate vessel dedicated to the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 10^4 cells/ml.

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1.
- Source (laboratory, culture collection): In-house laboratory culture
- Pre-culture (at test initiation): 3 or 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10E4 cells/ml.
- Pre-culture medium: M2; according to the OECD 201 Guideline, formulated using Milli-Q water (tap water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges: Milli-Q water; Millipore Corp., Bedford, Mass., USA) preventing precipitation and with the following composition:
NH4Cl 15 mg/l
MgCl2.6H2O 12 mg/l
CaCl2.2H2O 18 mg/l
MgSO4.7H2O 15 mg/l
KH2PO4 1.6 mg/l
FeCl3.6H2O 64 µg/l
Na2EDTA.2H2O 100 µg/l
H3BO3 185 µg/l
MnCl2.4H2O 415 µg/l
ZnCl2 3 µg/l
CoCl2.6H2O 1.5 µg/l
CuCl2.2H2O 0.01 µg/l
Na2MoO4.2H2O 7 µg/l
NaHCO3 50 mg/l

pH 8.1 ± 0.2

- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm) in a climate room at a temperature of 21-24°C.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol/l (24 mg CaCO3/l)

Test temperature:

between 22.1 and 22.9°C

pH:

between 8.0 and 8.3

Nominal and measured concentrations:

Nominal: 0, 100 mg/l
Measured: Analysis of the samples taken during the limit test showed that measured concentrations were in agreement with nominal at the start (102%) and remained stable during the first 24 hours of exposure (99% of nominal). However, a slight decrease was observed at the end of the test (71% of nominal). Given these results, effect parameters were based on the time weighted average exposure concentration that was calculated to be 89 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml, all-glass, containing 50 ml of test solution
- Type (delete if not applicable): closed
- Aeration: no
- Initial cells density: 10000 cells/ml
- Mean control end cells density: 1813000 cells/ml
6 replicates of both the control and the limit concentration;
1 extra replicate of both the control and the limit concentration for sampling purposes;
2 replicates of the limit concentration without algae.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- test medium: M2 (according to the OECD 201 Guideline)
- Source/preparation of dilution water: tap water purified by Reverse Osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges: Milli-Q water; Millipore Corp., Bedford, Mass., USA) preventing precipitation
- the following chemicals (analytical grade) are dissolved in the purified tap water:
NH4Cl 15 mg/l
MgCl2.6H2O 12 mg/l
CaCl2.2H2O 18 mg/l
MgSO4.7H2O 15 mg/l
KH2PO4 1.6 mg/l
FeCl3.6H2O 64 µg/l
Na2EDTA.2H2O 100 µg/l
H3BO3 185 µg/l
MnCl2.4H2O 415 µg/l
ZnCl2 3 µg/l
CoCl2.6H2O 1.5 µg/l
CuCl2.2H2O 0.01 µg/l
Na2MoO4.2H2O 7 µg/l
NaHCO3 50 mg/l
- pH 8.1 ± 0.2
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 81 to 93 micro-E.m^2.s^1.
- Incubation: Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- 72 h NOErC, 72 h NOEyC, 72 h ErC50, 72 h EyC50

TEST CONCENTRATIONS
Range finding study:
- Test concentrations: 0, 0.1, 1, 10, 100 mg/l
- Results used to determine the conditions for the definitive study: EC50
Final test:
- Test concentrations: 0, 100 mg/l

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: confirmed by analysis

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 89 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: confirmed by analysis

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 89 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes (for growth curve see attached background material)
- Observation of abnormalities (for algal test): no
- Any stimulation of growth found in any treatment: no
- Other: relatively low growth rate during the first 24 hours of exposure that was observed in both the control and the limit group, yet at the end of the test in both groups cell densities could safely be compared to each other
No significant differences were recorded between the values for growth rate or yield at the limit concentration when compared to the control group.

Results with reference substance (positive control):

The EC50 for growth rate reduction (ERC50: 0-72h) was 1.4 mg/l with a 95% confidence interval ranging from 1.1 to 1.9 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ERC50: 0-72h for the algal culture tested corresponds with this range.

The EC50 for yield inhibition (EYC50: 0-72h) was 0.56 mg/l with a 95% confidence interval ranging from 0.34 to 0.94 mg/l. The historical ranges of the 72h-EC50 for yield inhibition lie between 0.43 and 1.1 mg/l. Hence, the EYC50: 0-72h for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

Statistical analysis of the data was not needed as the effects recorded were not significant (<10%).
No EC50-values could be calculated because the test substance proved to be non-toxic (EC50 > maximum concentration tested).

Any other information on results incl. tables

The average exposure concentration is considered to be equivalent to the Time Weighted Average (TWA) of the concentrations of Neosolue-Aqulio measured in the samples taken at the start (C_t=0), after 24 hours (C_t=24) and the end of the test (C_t=72). (for calculation formula see attached background material)

During the range-finding test period, no effects on growth rate and yield were observed at any of the test concentrations and the control. The expected EC₅₀for both growth rate reduction and yield inhibition was above the nominal concentration of 100 mg/l.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the described test Neosolue-Aqulio did not induce growth rate reduction or yield inhibition of Pseudokirchneriella subcapitata at 89 mg/l after 72 hours of exposure (NOEC). The EC50 for both growth rate reduction (ERC50: 0-72h) and yield inhibition (EYC50: 0-72h) was >100 mg/l.

It is noted that the EC50 based on measured (TWA) concentrations actually is >89 mg/L (max concentration tested). However because at 89 mg/L no adverse effects were observed (NOEC=89 mg/L) it can be concluded that the EC50 will also be >100 mg/L and therefore the EC50 is reported as >100 mg/L.