Hexyl benzoate

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:

15 September 2017 - 30 November 2017

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:

23 March 2006; Annex 5 corrected 28 July 2011

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

Version / remarks:

14 December 2000

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Single samples were taken from all test concentrations and the control according to the schedule below.

Frequency: at t=0, 24, 48 and 72 h
Volume: 2.0 mL*
Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

At the end of the exposure period, the replicates with algae were not pooled at each concentration before sampling.

Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration (10% saturated solution) but without algae. Samples for analysis were taken at the start of the test period and after 24, 48 and 72 hours of exposure.

*Sampling after 24 hours of exposure in the final test was performed with an uncalibrated syringe and the sampling volume might have slightly deviated from the volume of 2.0 mL. This may have had an impact on the measured concentrations. Considering the relatively quick decrease of exposure concentrations, however, it was considered that the eventual possible impact on the determined average exposure concentrations was negligible.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Correction for purity: no correction for purity was made.
- Method: Direct application to the test medium.
The substance was a colourless liquid and not completely soluble in test medium at the loading rate initially prepared. Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of 190-197 minutes. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
- Based on information provided by the Sponsor, the test item was treated as being volatile, and therefore the risk of losses by volatilization of the test item from the aqueous media was kept to a minimum by having no headspace.
- Controls: test medium without test item or other additives.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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 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, in the range of 400-700 nm) in a climate room at a temperature of 21-24°C.

CULTURING
- Pre-culture:
3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (Adjusted M2 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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

21 - 23°C throughout the test.

pH:

7.4 - 8.0 throughout the test.

Nominal and measured concentrations:

Nominal concentrations: 10, 18, 32, 56 and 100% of a saturated solution prepared at a nominal loading rate of 100 mg/L
Mean exposure concentrations: 0.0088, 0.011, 0.016, 0.10 and 0.44 mg/L, respectively corresponding with nominal concentrations stated above. These concentrations are the geometric mean exposure concentrations for the first 48 hours of exposure. See 'Any other information on materials and methods' for the calculation of the geometric mean exposure concentrations.
For measured concentrations at individual sampling times, see table 1 in 'Any other information on results'.

Details on test conditions:

TEST SYSTEM
- Test vessel: 40 mL, air-tightly closed vessels due to volatility of the substance; fill volume: 40 mL.
- Initial cells density: 1 x 10^4 cells per mL
- Control end cells density: 31.4 x 10^4 cells/mL (at 48 h), 187.1 x 10^4 cells/mL (at 72 h)
- No. of vessels per concentration (replicates): 3 (+ 1 extra replicates of each test concentration for sampling purposes)
- No. of vessels per control (replicates): 5 (Initially, 6 control vessels were tested. However, one test vessel broke after the cell density measurement at 48 hours of exposure. This was taken into consideration during the analysis of the test results but was determined to have no impact on the interpretation of the results after 72 h of exposure).
- Other: 1 or 2 extra replicates of each test concentration without algae

GROWTH MEDIUM
- Stock culture medium: M1 prepared 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 (prepared in accordance with OECD 201 using reverse osmosis purified deionised water (Milli-RO, Millipore), with 300 mg/L NaHCO3 and 6 mmol/L HEPES buffer.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 72 to 75 µE/m2/s.
- During the incubation, the algal cells were kept in suspension by continuous shaking.
- Intervals of water quality measurements: pH: at the beginning and at the end of the test. Temperature of medium: continuously in a temperature control vessel.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm) in the combined limit/range-finding test and with cuvettes (path length = 10 mm) in the final test. Algal medium was used as blank and the extra replicates as background for the treated solutions.
- Appearance of the cells: At the end of the final test microscopic observations were performed on the algae in the control and the 32% SS test group to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 1.0, 10 and 100% of a saturated solution prepared at 100 mg/L
- Results used to determine the conditions for the definitive study: yes, algal growth was slightly inhibited at all concentrations (=< 6.2% inhibition). Test concentrations were below the detection limit after 24 hours of exposure and in consultation with the sponsor it was decided to perform the final test as a full test and use air-tight closed vessels considering the possible volatility of the substance. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the highest test concentration when compared to the control.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (September 2017)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.41 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95%-CI: 0.33-0.54 mg/L

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities in algal cells: Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 0.016 mg/L when compared to the control.
- pH and temperature were maintained within the limits prescribed by the study plan.

- In the final test, exposure concentrations decreased quickly and were below the limit of detection from 24 hours onwards at the three lowest test concentrations, and from 48 hours onwards for all but the highest test concentration. At the end of the test, all test concentrations had decreased below the limit of detection. Consequently, it was not possible to accurately calculate exposure concentrations representing actual exposure of the algal cells throughout the 72-hour test period. Therefore, the biological data were assessed to determine if the validity criteria of the test guideline were met for the first 48 hours of exposure, as effects on algal growth were considered not to further increase after 48 hours while exposure concentrations could be more reliably estimated for the first 48 hours. As the validity criteria were met, it was decided to derive the ErC50 in this study for the first 48 hours of exposure. The EC10, EC20 and NOEC could not be derived because of the above mentioned decrease in test concentrations.

- Inhibition of growth rate was considerably higher in the final test compared to the combined limit/dose-range finding study (see table 2 and table 3). This was likely due to the difference in test vessels used (air-tight in the final test compared to open vessels in the combined limit/dose-rang finding study) and therefore the amount of substance to which the algae were exposed.

- Analysis showed that the lower concentrations decreased below the limit of detection within 24 hours of exposure, while the concentrations measured in 56 and 100% SS decreased below the limit of detection after 48 and 72 hours of exposure, respectively. The concentrations measured in the samples without algae decreased slightly faster but were comparable at the start of the test.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 72h-ErC50: 1.1 mg/L (95% confidence interval ranging from 1.1 to 1.1 mg/L)
- Other: The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ErC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

Statistically significant effects: Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller
Calculation of EC50 values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition versus the logarithms of the corresponding average exposure concentrations of the substance.

The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Table 1 Measured concentrations versus nominal concentrations

Substance % SS prep. at 100 mg/L	Measured concentration (mg/L)			Average conc.(mg/L)
	t=0h	t=24h	t=48h
10	0.281	0.0016^#	0.0016^#	0.0088
18	0.485	0.0016^#	0.0016^#	0.011
32	0.830	0.0034	0.0016^#	0.016
32^*	0.893	0.0016^#	0.0016^#	0.013
56	1.48	0.459	0.0016^#	0.10
100	2.70	1.46	0.022	0.44

^*without algae; ^# Estimated as a factor of 2 below the limit of detection.

Table 2 Growth rate and percentage inhibition in the combined limit/range-finding test

Substance % SS prep. at 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	1.921	0.0157	6
1.0	1.894	0.0329	3	1.4
10	1.894	0.0205	3	1.4
100	1.802	0.0412	6	6.2

Table 3 Growth rate and percentage inhibition in the final test

Substance % SS prep. at 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	1.719	0.0310	6
10	1.695	0.1248	3	1.4
18	1.625	0.0336	3	5.5
32	1.463	0.0616	3	15
56	1.184	0.0626	3	31
100 (0.44)	0.863	0.0059	3	50

 ( ) The corresponding average concentration is reported between brackets in mg/L.

Table 4 Growth rate and percentage inhibition in the final test at different time intervals

Substance % SS prep. at 100 mg/L	n	0 – 24 h		24 – 48 h
		Mean	%Inhibition	Mean	%Inhibition
Control	6	1.810		1.627
10	3	1.872	-3.4	1.517	6.8
18	3	1.873	-3.5	1.376	15
32	3	1.875	-3.6	1.051	35
56	3	1.541	15	0.827	49
100 (0.44)	3	1.408	22	0.319	80

( ) The corresponding average concentration is reported between brackets in mg/L.

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks'

Conclusions:

The EC50 for growth rate inhibition (48h-ERC50) was 0.41 mg/L, based on average concentrations, with a 95% confidence interval ranging from 0.33 to 0.54 mg/L.

Executive summary:

A study was performed to assess the effect of the substance on the growth of the green alga Pseudokirchneriella subcapitata. The test was performed according to OECD test guideline 201 and under GLP principles. A Saturated Solution (SS) was prepared at a nominal loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration and were 10, 18, 32 and 56% of the SS. The final test was performed using exponentially growing algal cultures which were exposed for a total of 72 hours to an untreated control (five replicates) and three replicates per test concentration. The study was conducted using air-tight vessels due to likely volatility of the substance.

 

Samples for analytical confirmation of actual exposure concentrations were taken at t=0, 24, 48 and 72 h and analysed with a validated HPLC-UV method. The lower concentrations decreased below the limit of detection within 24 hours of exposure, while the concentrations measured in 56 and 100% SS decreased below the limit of detection after 48 and 72 hours of exposure, respectively.

The effect parameters were eventually based on the results obtained after 48 hours of exposure because it was not possible to accurately calculate exposure concentrations representing actual exposure of the algal cells throughout the 72 -hour test period. The geometric mean exposure concentrations were calculated to be 0.0088, 0.011, 0.016, 0.10 and 0.44 mg/L in 10, 18, 32, 56 and 100% of the SS prepared at 100 mg/L, respectively, over the test period of 48 hours. All acceptability criteria were met and the study was considered reliable.

The 48h-ErC50 was calculated to be 0.41 mg/L, based on geometric mean concentrations (95% confidence interval: 0.33 -0.53 mg/L). No ErC10 or NOEC values could be derived since the exposure concentrations decreased quickly and were below the limit of detection from 24 h onwards at the three lowest test concentrations, and from 48 h onwards for all but the highest test concentrations.

Description of key information

The 48h-ErC50 of the substance towards algae was 0.41 mg/L, based on geometric mean exposure concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.41 mg/L

Additional information

A study was performed to assess the effect of the substance on the growth of the green algaPseudokirchneriella subcapitata.The test was performed according to OECD test guideline 201 and under GLP principles. A Saturated Solution (SS) was prepared at a nominal loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration and were 10, 18, 32 and 56% of the SS. The final test was performed using exponentially growing algal cultures which were exposed for a total of 72 hours to an untreated control (five replicates) and three replicates per test concentration. The study was conducted using air-tight vessels due to likely volatility of the substance.

 

Samples for analytical confirmation of actual exposure concentrations were taken at t=0, 24, 48 and 72 h and analysed with a validated HPLC-UV method. The lower concentrations decreased below the limit of detection within 24 hours of exposure, while the concentrations measured in 56 and 100% SS decreased below the limit of detection after 48 and 72 hours of exposure, respectively.

The effect parameters were eventually based on the results obtained after 48 hours of exposure because it was not possible to accurately calculate exposure concentrations representing actual exposure of the algal cells throughout the 72 -hour test period. The geometric mean exposure concentrations were calculated to be 0.0088, 0.011, 0.016, 0.10 and 0.44 mg/L in 10, 18, 32, 56 and 100% of the SS prepared at 100 mg/L, respectively, over the test period of 48 hours.

All acceptability criteria were met and the study was considered reliable.

The 48h-ErC50 was calculated to be 0.41 mg/L, based on geometric mean concentrations (95% confidence interval: 0.33 -0.53 mg/L). No ErC10 or NOEC values could be derived since the exposure concentrations decreased quickly and were below the limit of detection from 24 h onwards at the three lowest test concentrations, and from 48 h onwards for all but the highest test concentrations.