Benzyl salicylate

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:

2003-02-14 to 2003-06-03

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Nominal concentrations of benzyl salicylate were 0 mg/L (control), 0.50, 1.0, 2.0, 4.0, and 8.0 mg/L. Initial measured concentrations of benzyl salicylate were: ND (none detected at or above the limit of quantitation; control), 0.502, 1.01, 1.92, 3.85, and 7.39 mg/L. Final measured concentrations ranged from 4 to 68% of nominal concentrations. Insoluble material was not observed at any tested concentration during the tes.

Vehicle:

no

Details on test solutions:

It was performed at 24 ± 2°C with five concentrations of test substance and a control. An 8.0 mg/L stock solution was prepared on April 3, 2003 by weighing 0.0160 g of test substance into a 2,000 mL Class A glass volumetric flask and adjusting the volume of dilution water to the line. This stock solution was mixed overnight on a magnetic stirrer at room temperature. A series of solutions was prepared on April 4, 2003, by bringing 63, 130, 250, 500, and 1,000 mL of the 8.0 mg/L stock solution to 1,000 mL with dilution water. Nominal concentrations of benzyl salicylate were 0 mg/L (control), 0.50, 1.0, 2.0, 4.0, and 8.0 mg/L. A portion of each solution was transferred into a 1.0 L glass beaker and a 1.0 L portion of dilution water was also transferred to a glass beaker to serve as a control.

Test organisms (species):

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

Details on test organisms:

Algae used for the test (Selenastrum capricornutum, UTEX 1648) were from a culture originally procured from the Culture Collection of Algae at the University of Texas at Austin and delivered to T.R. Wilbury Laboratories on July 17, 2001. The culture was transferred to sterile enriched media identical to media used for this test and maintained at test conditions for at least 14 days before the definitive test.

During the acclimation period, the culture was actively growing in at least 2 subcultures prior to the start of the toxicity test. The subsample of algae used to inoculate media at the start of the definitive test came from an eight day old culture. Identification of the culture organisms, which are also referred to as Raphidocelis subcapitata and Pseudokirchneriella subcapitata, was verified using an appropriate taxonomic key.

Test type:

static

Water media type:

freshwater

Remarks:

Water used for acclimation of test organisms and for all toxicity testing was sterile freshwater AAP medium at a pH of 7.5 ± 0.1.

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

A 0.5 mL aliquot of test media from each test vessel where growth was maximally inhibited (3.85 and 7.39 mg/L benzyl salicylate concentrations) was combined in a 250-mL flask with 100 mL of fresh media to determine whether toxic effects were algicidal or algistatic. This culture was incubated under test conditions for 168 hours.

Hardness:

No data.

Test temperature:

Temperature range during the 72 hour incubation : 24.0 to 24.7 °C.

pH:

The pH of the test solutions was measured at Time 0 and after 72 hours of incubation :

Test Concentration pH at Time 0 pH at 72 hours
Control 7.5 9.3 - 9.7
0.50 mg/L (nominal) 7.5 9.7 - 9.8
1.0 mg/L (nominal) 7.5 8.5
2.0 mg/L (nominal) 7.4 7.5
4.0 mg/L (nominal) 7.4 7.4
8.0 mg/L (nominal) 7.4 7.4

Dissolved oxygen:

No data.

Salinity:

No data.

Nominal and measured concentrations:

Nominal concentrations: Control, 0.5, 1.0, 2.0, 4.0 and 8.0 mg/L
Initial measured concentration: Not Detected, 0.502, 1.01, 1.92, 3.85 and 7.39 mg/L
Measured concentrations after 72hours: Not Detected, 0.0249, 0.340, 1.18, 2.71, 5.19 mg/L

Details on test conditions:

Vessels sealed with minimum head space.

Solutions were subdivided into 11 clear glass 40 mL vials for each treatment (the control was subdivided into 20 replicates) and the vials, which were filled to capacity to eliminate any head space, were sealed with Teflon-lined caps. Test vessels were randomly arranged on a rotary shaker adjusted to approximately 100 rpm in an incubator during the test (a random numbers table was used to select the location for each vessel). A 24 hour light and 0 hour dark photoperiod was automatically maintained with cool-white fluorescent lights that provided a light intensity of approximately 390 footcandles (approximately 55 to 56 μEin/m2sec).
The number of algal cells/mL in each test vessel and the occurrence of relative size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells was determined visually by means of direct microscopic examination with a hemocytometer. At 24, 48, and 72 hours, three treatment vessels and six control vessels were randomly selected and sacrificed (opened to the atmosphere) to allow daily determination of the number of algal cells/mL. The remaining two vessels at each concentration were used for the determination of benzyl salicylate concentration at the end of the test.
Temperature of the incubator was measured and recorded daily (thermometer number 2968) and the temperature in a representative vessel of water incubated with the test vessels was continuously recorded. The pH of test solutions was measured and recorded in the single solution of each concentration prior to its distribution to test vessels at the beginning of the test, and in all test vessels used for the determination of the number of algal cells/mL at the end of the test.

A 0.5 mL aliquot of test media from each test vessel where growth was maximally inhibited (3.85 and 7.39 mg/L benzyl salicylate concentrations) was combined in a 250-mL flask with 100 mL of fresh media to determine whether toxic effects were algicidal or algistatic. This culture was incubated under test conditions for 168 hours.

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.691 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI : 0.628 - 0.759 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.502 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.29 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI : 1.18 - 1.41 mg/L

Details on results:

Insoluble material was not observed during the test. Nominal concentrations of benzyl salicylate were: 0 mg/L (control), 0.50, 1.0, 2.0, 4.0, and 8.0 mg/L. Initial measured concentrations of benzyl salicylate were: ND (none detected at or above the limit of quantitation; control), 0.502, 1.01, 1.92, 3.85, and 7.39 mg/L. These initial measured concentrations ranged from 92 to 101% of nominal concentrations. Final measured concentrations were 4 to 68% of the nominal measured concentrations, indicating that once the aqueous solutions of benzyl salicylate were sealed into the test vessels with the algae, concentrations decreased during the 72-hour exposure period.

The algal population grew at an acceptable rate in the sealed vessels with no head space, resulting in an average of 266,000 cells/mL in the control after 72 hours. No effects (relative size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells) were noted during the test. Water quality throughout the test was within acceptable limits. The incubator temperature ranged from 24.0 to 24.7°C. The pH was not affected by the test substance.

The 24, 48, and 72 hour EC50 values are presented in Table 7. Exposure of Selenastrum capricornutum to benzyl salicylate for 72 hours resulted in an EC50 of 0.691 mg/L when calculated using the average specific growth rate, 0.735 mg/L when calculated using the number of cells/mL, and 1.29 mg/L when calculated using the area under the growth curve. The 72 hour NOEC is 0.502 mg/L benzyl salicylate when determined using the number of cells/mL or the average specific growth rate, at <0.502 mg/L when determined using the area under the growth curve.

At the conclusion of the definitive toxicity test, a 0.5 mL aliquot of test media from each test vessel where growth was maximally inhibited (the 3.85 mg/L and 7.39 mg/L benzyl salicylate concentrations) was combined with 100 mL of fresh media in a 250-mL flask. These cultures were incubated under test conditions for 168 hours. During this period the number of algal cells increased from an initial calculated concentration of <10,000 cells/mL to 828,000 cells/mL at 3.85 mg/L and 274,000 cells/mL at 7.39 mg/L, indicating that the toxic effects were algistatic rather than algicidal.

Results with reference substance (positive control):

10,000 (0h) to 266,000 (72h), i.e. factor of 266, however does not appear to be in exponential growth phase from 0 to 24 h, only 24 to 48 to 72 hr.

Reported statistics and error estimates:

No data.

Initial measured concentrations were 92- 100 % of nominal compared to final which were 4- 68 % nominal. This decrease is considered to be due to adsorption to the increasing algal biomass and not loss of substance from the test system as sealed vessels were used with minimum headspace. Furthermore, the substance was shown to be stable in test medium over the test period (106% of nominal after 72 hours).

Validity criteria fulfilled:

not specified

Conclusions:

The toxicity of the test substance to Freshwater Alga was assessed according to OECD guideline 201. The NOErC was found to be 0.502 mg/L (growth rate, number of cells) and ErC50(72h) = 1.29mg/L.

Description of key information

In an OECD 201 algal inhibition test, Benzyl salicylate was found to be acutely toxic to algae: ErC50(72h) = 1.29mg/L, NOEC(72h) = 0.502mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

1.29 mg/L

EC10 or NOEC for freshwater algae:

0.502 mg/L

Additional information

The preferred observational end point in this study is algal growth rate inhibition because it is not dependent on the test design (ECHA guidance Chapter R.7b v1.1). Thus the 72-h EC50 and NOEC based on growth rate are used for classification purposes.