Gum benzoin, Siam

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:

07 Jan 2018 - 16 Feb 2018

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:

Adopted March 23, 2006; Annex 5 corrected 28 July 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Solubility in water: 43593 mg/L at 24°C for the main constituent (benzoic acid)
Stability in water: Somewhat stable
Vapour pressure: 1.4 Pa

Analytical monitoring:

yes

Details on sampling:

- Concentrations: WAFs individually prepared at loading rates of 4.6, 10, 22, 46 and 100 mg/L
- Sampling method: 40 mL samples for possible analysis were taken from all test concentrations and the control at t=0 h, t=24 h, t=48h and t=72 h.
- Sample storage conditions before analysis: Samples were stored in a refrigerator (2-8°C) until analysis.
- At the end of the exposure period, the replicates with algae were not pooled at each concentration before sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item was heated in the oven at 75ºC for 24 hours prior to weighing. The container was closed during heating.
Preparation of test solutions started with loading rates individually prepared ranging between 1.0 and 100 mg/L. Exact amounts of test item were added to test medium containing no HEPES buffer. A one-hour treatment period of ultrasonic waves was applied and was followed by two days of magnetic stirring to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure of the second full test. WAFs prepared at a loading rate of 100 mg/L in the range-finding and first full test were clear and slightly yellow.
After preparation, volumes of 40 mL were added to each replicate of the respective test concentration. Subsequently, 0.8 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL and HEPES buffer (0.24 mL) was spiked to each vessel.
Solutions for analysis of TOC concentrations:
• At the start of the test: samples were taken from freshly prepared solutions (before preparation of the exposure vessels).
• At the end of the exposure period: volumes of 40 mL of each WAFs were added to vessels with no algae at the start of the test and used as solutions for analysis of TOC concentrations at the end of the test. These vessels were not spiked with HEPES buffer. This was done to prevent that the carbon originating from the buffer will obscure the results of TOC analysis.
- Controls: Test medium without test item or other additives

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata)
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum (at test initiation): 3 days
- 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 in a climate room at a temperature of 21-24°C.
- 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.
- Stock culture medium: M1, according to NPR 6505
- Pre-culture and test medium: Adjusted M2, according to OECD 201, formulated using Milli-RO water, with 6 mmol/L HEPES buffer and 300 mg/L NaHCO3.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

22 - 24°C

pH:

At t=0 h: 7.2-7.3
At t=72 h: 7.8-8.0

Nominal and measured concentrations:

Range finding test:
Nominal: loading rates of 10 and 100 mg/L.
Measured concentrations (t=0h):0.51 and 8.8 mg TOC/L
Measured concentrations (t=72h): 0.25 and 3.0 mg TOC/L

Full test:
Nominal: Control, WAFs individually prepared at loading rates of 4.6, 10, 22, 46 and 100 mg/L
Measured concentrations (t=0h): 0.11, n.q, n.q, n.q, 0.97, n.q
Measured concentrations (t=72h): n.q, n.q, n.q, n.q, 1.4, n.q

n.q. not quantifiable.

Details on test conditions:

TEST SYSTEM
- Test vessel: 40 mL, all-glass vials containing 40 mL of test solutions, airtight closed without headspace.
- Aeration: no
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 155 x 10^4 cells/mL
- 3 replicates of each test concentration
- 6 replicates of the control
- 6 extra replicates of each test group without algae and HEPES buffer for sampling purposes after 24, 48 and 72 hours of exposure
- 1 replicate of each test group without algae used to correct for the background of the treatments during measurements of the cell densities

GROWTH MEDIUM
- Standard medium used: M2 medium adjusted with HEPES buffer and additional NaHCO3.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: adjusted M2 medium, formulated using Milli-RO water (tap-water purified by reverse osmosis)
- Total organic carbon: 0.11 mg TOC/L measured at t=0h, not quantifiable at t=24, 48 and 72 h.
- Intervals of water quality measurement: pH: at the beginning and at the end of the test, for the control and all test concentrations. Temperature of medium: continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod, light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 80 to 81 µE.m-2.s-1.

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 cuvettes (path length =10 mm) at 24-hour intervals. Adjusted M2 medium was used as blank and the extra replicates, without algae, as background for the treated solutions.

TEST CONCENTRATIONS
- Range finding study test concentrations: WAFs individually prepared at loading rates of 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (performed Jan 2018)

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Based on biological relevance

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Based on statistical significance

Details on results:

Two full tests were performed based on the results of a preceding range-finding test. A first full test was considered not valid as the mean coefficient of variation for section-by-section specific growth rates in the control cultures exceeded 35%. Besides, the growth factor in the control treatment was two times lower than in the lowest loading rate tested. Only the results of the second full test has been recorded in this entry.

There was a clear difference in the measured TOC concentrations between the range-finding and the full test, whereas the difference between the biological results, although present, was not so striking.

The test item is a UVCB and poorly soluble substance for which water/medium solubility is unknown. Therefore, no conclusions regarding measured concentrations can be drawn. It can be possible that the actual solubility is below the limit of quantification of the TOC method. If it is the case, then the concentrations measured in the range-finding test resulted from oversaturation of WAFs. Nevertheless, it can be claimed that WAFs were prepared properly as an increase of effects with the applied dose was observed.

During the range-finding test, a concentration of 0.51 mg TOC/L measured in WAF of 10 mg/L caused a stimulation of growth of 2.6%. In contrary, during the final test an inhibition of 5.7% was observed in WAF of 100 mg/L in which concentrations were below the limit of detection. It seems that component(s) responsible for toxic effects were dissolved at concentration that could not be detected with the TOC method or do not contain organic carbon. For these reasons, the possibility that the higher effects observed in the range-finding test were due to oversaturation cannot be excluded.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the WAF prepared at a loading rate of 100 mg/L when compared to the control.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: The EC50 for growth rate inhibition (72h-ErC50) was 0.86 mg/L with a 95% confidence interval ranging from 0.84 to 0.88 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. The observed 72h-ErC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

Williams Multiple Sequential t-Test Procedure, α=0.05, one-sided, smaller (for determination of NOEL); calculations for ELx-values were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany). No EL10 for growth rate could be calculated since the effects recorded at the maximum
loading rate tested were below 10%. No EL50-values for both growth rate and yield inhibition could be calculated because the test
item proved to be non-toxic (EL50 > maximum loading rate tested).

Measured TOC concentrations

Actual concentrations could only be quantified at the WAF prepared at a loading rate of 46 mg/L. In the remaining groups the concentrations were either not detected or were lower than 1.0 mg/L.

Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared.

Growth Rate ( µ, day^-1) And Percentage Inhibition For The Total Test Period

Benzoin resoid Loading rate (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.680	0.0118	6
4.6	1.646	0.0129	3	2.0
10	1.685	0.0188	3	-0.31
22	1.643	0.0057	3	2.2^#
46	1.635	0.0132	3	2.7^#
100	1.584	0.0146	3	5.7^#

^#- effect was statistically significant however not biologically relevant (<10%)

Yield (x10⁴Cells/mL) And Percentage Inhibition For The Total Test Period

Benzoin resoid Loading rate (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	153.5	5.40	6
4.6	138.5	5.44	3	9.7
10	156.0	8.95	3	-1.7
22	137.4	2.36	3	10*
46	133.9	5.30	3	13*
100	115.0	5.05	3	25*

* - effect was statistically significant

Validity criteria fulfilled:

yes

Remarks:

In controls: cell density increased by an average factor of >16 within 72 hours, mean CV for section-by-section specific growth rates did not exceed 35% and CV of average specific growth rates during the whole test period did not exceed 7%

Conclusions:

The ErL50, ErL10 and NOEL for Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) exposed to Benzoin Resoid were >100, >100 and 10 mg/L (based on statistical significance).

Executive summary:

In a 72 h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to Water Accommodated Fractions (WAF) of the test substance individually prepared at loading rates of 4.6, 10, 22, 46 and 100 mg/L. Samples were analysed for Total Organic Carbon concentrations. Since the TOC-analysis is a non-specific method, the effect parameters were reported in terms of nominal loading rates initially prepared. Statistically significantly reduced growth rates were observed in algae exposed to WAFs prepared at 22, 46 and 100 mg/L, however these effects were considered not biologically relevant (<10%). The 72-hour ErL50 and ErL10 exceeded a loading rate of 100 mg/L.The 72h-NOEL for growth rate inhibition was 10 mg/L based on statistical significance and 100 mg/L based on biological relevance.

Description of key information

In a 72 h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to Water Accommodated Fractions (WAF) of the test substance individually prepared at loading rates of 4.6, 10, 22, 46 and 100 mg/L. Samples were analysed for Total Organic Carbon concentrations. Since the TOC-analysis is a non-specific method, the effect parameters were reported in terms of nominal loading rates initially prepared. Statistically significantly reduced growth rates were observed in algae exposed to WAFs prepared at 22, 46 and 100 mg/L, however these effects were considered not biologically relevant (<10%). The 72-hour ErL50 and ErL10 exceeded a loading rate of 100 mg/L.The 72h-NOEL for growth rate inhibition was 10 mg/L based on statistical significance and 100 mg/L based on biological relevance.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

72h-ErL50: > 100 mg/L

72h-ErL10: > 100 mg/L