Phenol, 2-methoxy-, reaction products with 2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane, hydrogenated

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:

From 30 november 2015 to 16 june 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Concentration of the test item in the test solution was determined at the beginning of the test and then at 24 hour intervals thereafter.
Analytical measurement was performed at the applied test concentration level and from the control at the beginning and at the end of the experiment.
The WAF samples were analysed by a GC-FID method, with quantification of four peak-groups for this UVCB substance.
Samples were taken (5 x 50 mL) in plastic tubes at the beginning of the test and then at 24 hour intervals at the applied test concentration level and from the control as well.
After sampling, WAF samples were frozen and kept approximately at -20°C at the Test Facility. One set of the samples (3 x 50 mL) were sent to the Test Site for analysis and one set (2 x 50 mL) were retained as a back-up at the Test Facility, if required for any confirmatory analyses (discarded after satisfactory results were obtained on the first set of).

Vehicle:

yes

Details on test solutions:

Because the test item is poorly soluble in water, test solution was prepared using a saturated solution method (water accommodated fraction, WAF) according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.
A saturated test item solution (nominal loading rate of 100 mg/L) was prepared by dispersing/dissolving the amount of test item into the test medium (OECD Medium) two days before the start of the study. This solution was shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C. The non-dissolved test material was removed by filtration through a fine (0.22 µm) filter to give the nominal loading rate of 100 mg/L.
The test substance is a UVCB, the above method is designed to maximise the concentration in solution of any fractions in the UVCB that may have some water solubility. The test is performed with a saturated solution.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd.
- Method of cultivation: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines.
The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, after an incubation period of three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

no data available

Test temperature:

Culture temperature was checked at the beginning of the experiment and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was in the range of 22.9 – 23.2 °C measured in the flask and between 22.7 and 23.6 °C measured within the climate chamber.

pH:

The pH was checked at the beginning and at the end of the test in each test vessels. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.24 – 8.75 during the experiment.

Dissolved oxygen:

no data available

Salinity:

no data available

Conductivity:

no data available

Nominal and measured concentrations:

Preliminary range-finding test : 0, 0.1, 1.0, 10.0 and 100.0 mg/ L nominal loading rate WAF
Because no significant inhibition of algal growth was observed during the preliminary range-finding test, only one test concentration of 100 mg/L nominal loading rate WAF and one control group were tested in a Limit Test.
Definitive test: 100.0 mg/ L nominal loading rate WAF

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile Erlenmeyer flasks
- Type (delete if not applicable): The flasks were covered with air-permeable stoppers.
- Material, size, headspace, fill volume: Volumes of 100 mL test solution per replicate in 250 mL sterile Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension.
- Aeration: The flasks were covered with air-permeable stoppers.
- Type of flow-through (e.g. peristaltic or proportional diluter): no available information
- Renewal rate of test solution (frequency/flow rate): no
- Initial cells density: approximately 10 exp 4 algal cells per mL test medium
- Control end cells density: 71.50 x 10 exp4 cell/ml
- No. of vessels per concentration (replicates): 11. Six replicates were used for observations and five replicates for the 24 and 48 hours analytical measurements.
- No. of vessels per control (replicates): 11. Six replicates were used for observations and five replicates for the 24 and 48 hours analytical measurements. Algal growth medium will be inoculated with algal cells (without test item) and examined in parallel to the test item concentrations.
- Because the test item is not stable over the 72h exposure period (based on the analytical method validation, Study Code: 15/064-901AN), study was extended to provide more data to allow a better interpretation.
Additional samplings for analysis at 24 hour intervals were used in order to better define loss of the test substance during the exposure period.


GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.
- Total organic carbon: no data available
- Particulate matter: no data available
- Metals: no data available
- Pesticides: no data available
- Chlorine: no data available
- Alkalinity: no data available
- Ca/mg ratio: no data available
- Conductivity: no data available
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: The pH was checked at the beginning and at the end of the test in each test vessels. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.24 – 8.75 during the experiment.
- Photoperiod: no. The algal culture flasks were continuously illuminated.
- Light intensity and quality: The light intensity at the position occupied by algal culture flasks during the test was about 7905 lux (equivalent to ~107 E/m2/s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed  15 % and therefore provided equal conditions for each test vessel.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study
- Test concentrations: A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations could be selected for use in the definitive test. Algal cells were exposed to each concentration of the test item plus a control, for 72 hours. The test was performed with two replicates per each test concentration and three replicates in the control group.
During the formulation procedure was prepared by the similar method described above, the required amounts of test item for the 100, 10 and 1 mg/L nominal loading rates WAFs were individually dispersed/dissolved into the test medium and handled as described above. For the lowest test concentration a dilution of the 10 mg/L nominal loading rate was prepared (0.1 % saturated solution of 10.0 mg/L nominal loading rate WAF).
The concentration levels used and results (72 h) of the Preliminary Range-Finding Test are summarised in the following table.
Table: Results of the Preliminary Range-Finding Test
Nominal concentrations
[mg/L nominal loading rate WAF] Untreated
control 0.1* 1.0 10.0 100.0
Average of cell number
at 72 hours (x 10exp4 cell/mL) 69.00 67.50 65.00 66.50 65.00
*Remark: 0.1 % saturated solution of 10.0 mg/L nominal loading rate WAF

- Results used to determine the conditions for the definitive study: Because no significant inhibition of algal growth was observed during the preliminary range-finding test, only one test concentration of 100 mg/L nominal loading rate WAF and one control group were tested in a Limit Test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.37 mg/L

Nominal / measured:

meas. (geom. mean)

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:

other: loading rate WAF

Basis for effect:

not specified

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.34 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

not specified

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate WAF

Basis for effect:

not specified

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.34 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

not specified

Details on results:

- Concentration of the test item: The test concentration was analytically determined at the start of the experiment and at 24 hour intervals thereafter. The corresponding measured geometric mean test item concentration was 0.34 mg/L.
The biological results are based on the nominal loading rate and on the measured geometric mean test item concentration.
- Average Specific Growth Rates: The results of the statistical evaluation (based on 2 Sample t-Test; alpha=0.05) show that the 0-72 h average specific growth rate was not statistically significantly different from the untreated control value, accordingly the No Observed Effect Concentration (NOEC) was determined as 100 mg/L loading rate WAF.
- AREAS UNDER THE Growth CURVES: The results of the statistical evaluation (based on 2 Sample t-Test; alpha=0.05) show that the 0-72 h areas were not statistically significantly different from the untreated control value, accordingly the No Observed Effect Concentration (NOEC) was determined as 100 mg/L loading rate WAF.
- YIELD : The results of the statistical evaluation (based on 2 Sample t-Test; alpha=0.05) show that the 0-72 h yield was not statistically significantly different from the untreated control value; accordingly the No Observed Effect Concentration (NOEC) was determined as 100 mg/L loading rate WAF.

Under the conditions of this algal growth inhibition test, a saturated solution had no effects on growth. The calculated endpoints for the effect of RHODIANTAL ORIGINAL IBCH were the following:
Based onthe nominal loading rates:
The 72h EbL50value (biomass):          >100 mg/L loading rate WAF
The 72h ErL50value (growth rate):    >100 mg/L loading rate WAF
The 72h EyL50value (yield):                >100 mg/L loading rate WAF
The72h No-Observed EffectLoading Rate (NOELR):                100 mg/L loading rate WAF
The72h Lowest Observed Effect Loading Rate (LOELR):         >100 mg/L loading rate WAF
Based on the measured geometric mean concentrations:
The 72h EbC50value (biomass):          >0.34 mg/L
The 72h ErC50value (growth rate):    >0.34 mg/L
The 72h EyC50value (yield):               >0.34 mg/L
The72h No-Observed Effect Concentration (NOEC):                  0.34 mg/L
The72h Lowest Observed Effect Concentration (LOEC):         >0.34 mg/L

Results with reference substance (positive control):

For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 16/281-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 13 – 16 September 2016.
The 72h ErC 50: 0.89 mg/L, (95 % confidence limits: 0.82 – 0.97 mg/L)
The 72h EbC 50: 0.62 mg/L, (95 % confidence limits: 0.57 – 0.67 mg/L)
The 72h EyC 50: 0.53 mg/L, (95 % confidence limits: 0.49 – 0.58 mg/L)

These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).

Reported statistics and error estimates:

The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period.
The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel 2007 for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
As a limit test was performed the EL50/EC50 values of the test item were not calculated and determined from the raw data.
Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (alpha = 0.05) by TOXSTAT software.
For the determination of the LOEC / LOELR and NOEC / NOELR, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by 2 Sample t-Test (alpha = 0.05) by TOXSTAT software.

Table 1: Calculation of exposure concentrations

Nominal loading rate WAF (mg/L)	Measured concentrations (mg/L)				Geometric mean (mg/L)
	start	24h	48h	72h/end
Control	n.d.	-	-	n.d.	-
100	0.50	0.38	0.35	0.19	0.34

 

n.d. – not detected

Table 2:    Growth Rates (µ) and Percentage Inhibition of µ during the Test Period

Test group	Growth rate µ and % inhibition of µ
	0–24 h		0–48 h		0–72 h
	µ	%	µ	%	µ	%
Control	0.0553	0.0	0.0605	0.0	0.0593	0.0
100 mg/L loading rate WAF	0.0553	0.0	0.0598	1.2	0.0591	0.3

 

Table 3:Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Test group	Areas under the Growth Curves (A) and % inhibition of A
	0–24 h		0–48 h		0–72 h
	A	%	A	%	A	%
Control	34.0	0.0	276.0	0.0	1330.0	0.0
100 mg/L loading rate WAF	34.0	0.0	268.0	2.9	1304.0	2.0

Table 4: Yield (Y) and Percentage Inhibition of Y during the Test Period

Test group	Yield (Y) and % inhibition of Y (0-72 h)
	Y	%
Control	70.5	0.0
100 mg/L loading rate WAF	69.7	1.2

 

Validity criteria fulfilled:

yes

Remarks:

see below

Conclusions:

Under the conditions of this algal growth inhibition test, a saturated solution had no effects on growth. The calculated endpoints for the effect of RHODIANTAL ORIGINAL IBCH were the following:
Based on the nominal loading rates:
The 72h EbL50 value (biomass): > 100 mg/L loading rate WAF
The 72h ErL50 value (growth rate): > 100 mg/L loading rate WAF
The 72h EyL50 value (yield): > 100 mg/L loading rate WAF
The 72h No-Observed Effect Loading Rate (NOELR): 100 mg/L loading rate WAF
The 72h Lowest Observed Effect Loading Rate (LOELR): > 100 mg/L loading rate WAF
Based on the measured geometric mean concentrations:
The 72h EbC50 value (biomass): > 0.34 mg/L
The 72h ErC50 value (growth rate): > 0.34 mg/L
The 72h EyC50 value (yield): > 0.34 mg/L
The 72h No-Observed Effect Concentration (NOEC): 0.34 mg/L
The 72h Lowest Observed Effect Concentration (LOEC): > 0.34 mg/L

The cell density in the control cultures increased by the factor of 71.50 within three days.
The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 13.84 %.
The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.61 %.
All validity criteria were met, therefore the study can be considered as valid.

Executive summary:

The effect of RHODIANTAL ORIGINAL IBCH test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours. Because no significant inhibition of algal growth was observed during the preliminary range-finding test, a Limit Test was carried out using only one test concentration of100 mg/L nominal loading rate WAFand one control group. The test concentration was analytically determined at the start of the experiment and at 24 hour intervals thereafter.The corresponding measured geometric mean test item concentration was 0.34 mg/L. The biological results are based on the nominal loading rate and on the measured geometric meantest item concentration. The test design included six replicates at test concentration and six replicates for the untreated control.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (a= 0.05) by TOXSTAT software.

The LC_{50 /}EC₅₀ values of the test item were determined directly from the raw data.

Under the conditions of this algal growth inhibition test, a saturated solution had no effects on growth. The calculated endpoints for the effect of RHODIANTAL ORIGINAL IBCH were the following:

Based onthe nominal loading rates:

The 72h E_bL₅₀value (biomass):          >100 mg/L loading rate WAF

The 72h E_rL₅₀value (growth rate):    >100 mg/L loading rate WAF

The 72h E_yL₅₀value (yield):                >100 mg/L loading rate WAF

The72h No-Observed EffectLoading Rate (NOELR):                100 mg/L loading rate WAF

The72h Lowest Observed Effect Loading Rate (LOELR):         >100 mg/L loading rate WAF

Based on the measured geometric mean concentrations:

The 72h E_bC₅₀value (biomass):          >0.34 mg/L

The 72h E_rC₅₀value (growth rate):    >0.34 mg/L

The 72h E_yC₅₀value (yield):               >0.34 mg/L

The72h No-Observed Effect Concentration (NOEC):                  0.34 mg/L

The72h Lowest Observed Effect Concentration (LOEC):         >0.34 mg/L

Description of key information

A saturated solution of RHODIANTAL ORIGINAL IBCH, had no effects on algal growth using the unicellular green algae Pseudokirchneriella subcapitata.

The 72-hour EL50 of RHODIANTAL ORIGINAL IBCH to Pseudokirchneriella subcapitata has determined to be > 100 mg/L (nominal loading rate WAF) based on the growth rate; revealing it is not harmful for the algae.

The 72h No-Observed Effect Loading Rate (NOELR):               100 mg/L loading rate WAF

The 72h Lowest Observed Effect Loading Rate (LOELR):       > 100 mg/L loading rate WAF

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

A GLP and OECD Guideline n°201 study, scored as Klimisch 1 and flagged as a key study, is available on Rhodiantal original ibch, giving a 72h-EL50 > 100 mg/L (nominal loading rate WAF) based on the growth rate; revealing it is not harmful for the algae.

The 72h No-Observed Effect Loading Rate (NOELR):               100 mg/L loading rate WAF

The 72h Lowest Observed Effect Loading Rate (LOELR):       > 100 mg/L loading rate WAF