Isobutyl 2-naphthyl ether

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 July 2016 - 02 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006 / Annex 5 corrected 28 July 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2008 / Amended by EC No. 2016/266 of 7 December 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Single 2-mL samples for possible analysis were taken from all test concentrations and the control at t=0 h, t=24 h and t=72. At the end of the exposure period, the replicates with algae were not pooled at each concentration before sampling. Instead, samples were taken from separate replicates. Samples were stored in a freezer until analysis.

Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate test item 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.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
The test item was a white solid and was heated to approximately 60°C during a period of about one hour prior to weighing. During this time, the test item turned into a clear liquid. No correction was made for the purity/composition of the test item. Preparation of test solutions started with a loading rate of 100 mg/L applying three days of magnetic stirring with minimum headspace followed by a settlement period of about two hours. Afterwards, the Saturated Solution (SS) was siphoned off and used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All final test solutions were clear and colourless. After preparation, volumes of 120 mL were added to each replicate of the respective test concentration. Subsequently, 2.4 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL. Controls contained test medium without test item or additives.

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: in-house laboratory culture
- Method of cultivation: algae stock cultures were started by inoculating growth medium (M1) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (intensity of 60 to 120 µE/m2/s in range λ=400-700 nm) in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Pre-culture: 3 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 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Pre-culture media and conditions: same as test medium, Adjusted M2

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

not indicated

Test temperature:

22 - 23°C

pH:

Start: 7.3 - 7.4
End: 7.5 - 8.0

Nominal and measured concentrations:

- Nominal test concentrations: 0.10, 0.32, 1.0, 3.2, 10, 32 and 100% of a saturated solution prepared at a loading rate of 100 mg/L (based on range-finding test).
- Average measured exposure concentrations: 0.0011, 0.0023, 0.0063, 0.026, 0.091, 0.32 and 1.1 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 120 mL, all-glass, minimum headspace, closed airtight, fill volume: 120 mL
- Initial cells density: 1 x 10^4 cells/mL
- Control end mean cells density: 130.1 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3 + 1 extra replicate of each test concentration for sampling purposes after 24 hours of exposure
- No. of vessels per control (replicates): 6
- No. of vessels without algae (replicates): 1 or 2 replicates of each test concentration

GROWTH MEDIUM
- Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
- Pre-culture and test medium: adjusted M2; according to the OECD 201 Guideline, formulated using Milli-RO

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Illumination: continuously using TLD-lamps with a light intensity within the range of 84 to 92 µE/m2/s

EFFECT PARAMETERS MEASURED: cell densities at 0, 24, 48 and 72 h.; pH was measured at the beginning and at the end of the test; temperature of medium was measured continuously in a temperature control vessel; appearance of the cells was determined at the end of the final test by microscopic observations on the control and the test groups contraining 10 and 32% of the SS to observe for any abnormal appearance of the algae.
- Determination of cell concentrations: at the beginning of the test, a counting chamber was used. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length = 10 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (July 2016)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.31 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.11 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.026 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- No observations of abnormalities

Growth rates were in the range of the controls at the three lowest test concentrations during the 72-hour test period, whereas the growth rate of algae exposed to 0.026 mg/L and higher were increasingly reduced. Inhibition of growth rate was statistically significant at the four highest test concentrations. The inhibition of 2.1% observed at 0.026 mg/L was considered biologically irrelevant (<10%) and the NOEC was therefore based on this concentration.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 72h-ErC50: 1.0 mg/L (95%-CI: 0.97-1.0 mg/L)
- Other: The observed 72h-ErC50 for the algal culture tested corresponds within the historical range for growth rate.

Reported statistics and error estimates:

- For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate (Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm, α=0.05, one-sided, smaller) or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).
- Calculation of ECx values was based on the cumulative distribution function (CDF) with the percentages of growth rate inhibition and the percentages of yield inhibition versus the corresponding TWA concentrations of the test item.
- The calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions®GmbH, Germany).

If detectable, the test concentrations decreased to 64 to 86% of the initial concentrations after 24 hours of exposure and to 11-57% of initial after 72 hours of exposure. The results were considered conclusive and the time weighted average (TWA) concentrations were calculated and used to determine the effects parameters (see Table 2). The range tested based on TWA concentrations was 0.0011, 0.0023, 0.0063, 0.026, 0.091, 0.32 and 1.1 mg/L. 

Table 1          Concentrations of the test item in test medium - final test

Time of sampling¹ [hours]	Percentage of SS^2 [%]	Analysed concentration [mg/L]	Relative to initial [%]
0	0	n.d.
	0.1	n.d.
	0.32	0.0042^4
	1.0	0.0153
	1.0^3	0.0149
	3.2	0.0554
	10	0.164
	32	0.502
	100	1.54
24	0	n.d.	n.a.
	0.1	n.d.	n.a.
	0.32	0.0029^4	70
	1.0	0.00993	65
	1.0^3	0.0127	86
	3.2	0.0405	73
	10	0.127	77
	32	0.323	64
	100	1.11	72
72	0	n.d.	n.a.
	0.1	n.d.	n.a.
	0.32	n.d.	n.a.
	1.0	n.d.	n.a.
	1.0^3	0.0075^4	50
	3.2	0.0061^4	11
	10	0.0327	20
	32	0.244	49
	100	0.887	57

¹      Samples were stored in the freezer (≤ -15°C) until the day of analysis.

²      Percentage of a saturated solution prepared at a loading rate of 100 mg/L.

³      Without algae.

⁴      Estimated value, calculated by extrapolation of the calibration curve

n.d. Not detected. The limit of detection of the method was determined to be 0.0021 mg/L taking a dilution factor of two into account.

n.a. Not applicable.

Table 2      Measured concentrations versus nominal concentrations (final test)

Naph Iso Butyl Ether Beta % SS prepared at 100 mg/L	Measured concentration (mg/L)			TWA (mg/L)
	t=0h	t=24h	t=72 h
0.10	0.00105^2	0.00105^2	0.00105^2	0.0011
0.32	0.00419	0.00292	0.00105^2	0.0023
1.0	0.0153	0.00993	0.00105^2	0.0063
1.0^1	0.0149	0.0127	0.00747	0.011
3.2	0.0554	0.0405	0.00607	0.026
10	0.164	0.127	0.0327	0.091
32	0.502	0.323	0.244	0.32
100	1.54	1.11	0.887	1.1

¹- without algae

²- LOD * 0.5

Table 3      Percentage inhibition of growth rate during the combined limit/range-finding test

Naph Iso Butyl Ether Beta % SS prepared at 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	1.713	0.0249	6
1.0	1.575	0.0227	3	8.1
10	1.048	0.0721	3	38.8
100	0.142	0.1450	6	91.7

Table 4       Percentage inhibition of growth rate (total test period) during the final test

Naph Iso Butyl Ether Beta TWA conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.623	0.0153	6
0.0011	1.667	0.0008	3	-2.7
0.0023	1.628	0.0164	3	-0.3
0.0063	1.628	0.0097	3	-0.4
0.026	1.589	0.0116	3	2.1*^#
0.091	1.477	0.0188	3	9.0*
0.32	0.809	0.0612	3	50.2*
1.1	0.021	0.0359	3	98.7*

* - effect was statistically significant

# - effect considered biologically irrelevant

VALIDITY OF THE TEST

1) In controls: cell denisity increased by an average factor of at least 16 within 72 hrs (i.e. 130).

2) Mean CV for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 9.7%).

3) CV of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (i.e. 0.9%)

Therefore the test is considered valid

Validity criteria fulfilled:

yes

Conclusions:

The ErC50, ErC10 and NOEC were 0.31, 0.11 and 0.026 mg/L respectively.

Executive summary:

A study was performed to assess the effect of the substance on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201. The test was performed under GLP. Preparation of test solutions started with a loading rate of 100 mg/L applying three days of magnetic stirring with minimum headspace followed by a settlement period of about two hours. Thereafter the Saturated Solution (SS) was siphoned off and used as the highest test concentration (100% of the SS). All lower test concentrations (0.10, 0.32, 1.0, 3.2, 10 and 32% of the SS) were prepared by subsequent dilutions of the SS in test medium. All final test solutions were clear and colourless. The test item was suspected to be volatile and hence testing was performed in closed air-tight vessels with minimum headspace and with adjusted medium. The algae were exposed for 72 hours (three replicate flasks per concentration, six replicate flasks per control). Samples were taken from all treatments at t = 0, 24 and 72 h and analysed with a validated UPLC-UV method. Time-weighted average concentrations were determined at 0.0011, 0.0023, 0.0063, 0.026, 0.091, 0.32 and 1.1 mg/L. The test concentrations from 1% SS onwards were ca 10-60% of the initial values. Below 1% SS the concentration were not detectable at the end. The validity criteria were met a.o., in the control the cell density increased by an average factor of at least 16 within 72 hrs (i.e. 130).The ErC50, ErC10 and NOEC were 0.31, 0.11 and 0.026 mg/L respectively, based on time-weighted average concentrations.

Description of key information

A study was performed to assess the effect of the substance on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201. The test was performed under GLP. Preparation of test solutions started with a loading rate of 100 mg/L applying three days of magnetic stirring with minimum headspace followed by a settlement period of about two hours. Thereafter the Saturated Solution (SS) was siphoned off and used as the highest test concentration (100% of the SS). All lower test concentrations (0.10, 0.32, 1.0, 3.2, 10 and 32% of the SS) were prepared by subsequent dilutions of the SS in test medium. All final test solutions were clear and colourless. The test item was suspected to be volatile and hence testing was performed in closed air-tight vessels with minimum headspace and with adjusted medium. The algae were exposed for 72 hours (three replicate flasks per concentration, six replicate flasks per control). Samples were taken from all treatments at t = 0, 24 and 72 h and analysed with a validated UPLC-UV method. Time-weighted average concentrations were determined at 0.0011, 0.0023, 0.0063, 0.026, 0.091, 0.32 and 1.1 mg/L. The test concentrationsfrom 1% SS onwards were ca 10-60% of the initial values.Below 1% SS the concentration were not detectable at the end.The validity criteria were met a.o., in the control the cell density increased by an average factor of at least 16 within 72 hrs (i.e. 130).The ErC50, ErC10 and NOEC were 0.31, 0.11 and 0.026 mg/L respectively, based on time-weighted average concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.31 mg/L

EC10 or NOEC for freshwater algae:

0.11 mg/L