4-tert-butylpyrocatechol

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 16-Sept-2010 to 27-Sept-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-tert-butylpyrocatechol

EC Number:

202-653-9

EC Name:

4-tert-butylpyrocatechol

Cas Number:

98-29-3

Molecular formula:

C10H14O2

IUPAC Name:

4-tert-butylbenzene-1,2-diol

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentrations of 4-TBC were analyzed in duplicate test media samples from the nominal concentrations of 0.25, 0.55, 1.20, 2.55, 5.55 and 12.0 mg/L.
- Sampling method: For measurement of the actual concentrations of 4-TBC, duplicate samples were taken daily from the test media of all test concentrations at the start of the test (without algae) and after 24, 48 and 72-hours (containing algae). At the same sampling times, duplicate samples were also taken from the control.
For the 24, 48 and 72-hour stability samples, additional flasks containing the test medium with algae were incubated for each treatment under the test conditions.
Two additional test concentrations of 0.55 and 5.55 mg/L without algae were incubated in parallel under the same test conditions as well as in the dark. Analytical samples were taken every 24 hours during the exposure period of 72 hours.
- Sample storage conditions before analysis: Immediately after sampling, the pH of each medium was adjusted to 6.0. All the samples were analyzed immediately after sampling.

Test solutions

Vehicle:

no

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: n° 61.81 SAG
- Source (laboratory, culture collection): The test organism used for the study was Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany).
- Age of inoculum (at test initiation): An inoculum culture was set up four days before the start of the exposure.
- Method of cultivation: The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines. The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.

ACCLIMATION
- Acclimation period: four days
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol/L (= 24 mg/L as CaCO3)

Test temperature:

The test flasks were incubated in a temperature-controlled water bath at a temperature between 22 and 23 °C.

pH:

At the start of the test, the pH measured in the treatments was 6.5. At the end of the test, pH values of 6.5 to 6.6 were measured.

Dissolved oxygen:

Not applicable

Salinity:

Not applicable

Nominal and measured concentrations:

nominal concentrations: 0, 0.25, 0.55, 1.20, 2.55, 5.55, 12.0 mg/L
measured concentrations: 0, 0.20, 0.46, 0.99, 2.10, 4.73, 10.8 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks were used.
- Type (delete if not applicable): closed; Each test flask was covered with a glass dish.
- Material, size, headspace, fill volume: The volume of test solution in each test flask was 15 mL.
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter): none (static test)
- Renewal rate of test solution (frequency/flow rate): a static, non-renewal exposure system was used.
- Initial cells density: 10000 cells/mL
- Control end cells density: The algal cell density in the pre-culture was determined by an electronic particle counter (Coulter Counter, Model ZM).
- No. of vessels per concentration (replicates): three replicates
- No. of vessels per control (replicates): six replicates

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water prepared according to the test guidelines was used for algal cultivation and testing. Analytical grade salts were dissolved in sterile purified water to obtain the following concentrations:

Macro-nutrients:
NaHCO3: 250.0 mg/L
KH2PO4: 1.6 mg/L
MgSO4 x 7 H2O: 15.0 mg/L
MgCl2 × 6 H2O: 12.0 mg/L
CaCl2 × 2 H2O: 18.0 mg/L
NH4Cl: 15.0 mg/L
Trace elements:
H3BO3: 185.0 µg/L
MnCl2 × 4 H2O: 415.0 µg/L
ZnCl2: 3.0 µg/L
CoCl2 × 6 H2O: 1.5 µg/L
CuCl2 × 2 H2O: 0.01 µg/L
Na2MoO4 × 2 H2O: 7.0 µg/L
FeCl3 × 6 H2O: 64.0 µg/L
Na2EDTA × 2 H2O: 100.0 µg/L

To keep the pH of the test media as constant as possible 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) were added to the test water.

- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH was measured and recorded in each treatment at the start and at the end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test media was also recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: yes. The pH of the test water was adjusted to 6.0 with a 1 M hydrochloric acid solution (the pH in the test media after addition of the algae was 6.5).
- Photoperiod: The test flasks were illuminated by fluorescent tubes (Philips TLD 36W/840), installed above the test flasks.
- Light intensity and quality: The mean measured light intensity at the level of the test solutions was approximately 7300 Lux (range: 6620 to 7660 Lux, measured at nine places in the experimental area). The light intensity was within ± 15% from the average light intensity as recommended by the guideline.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: A small volume of the algal suspension was withdrawn daily from each test flask for the measurement of the biomass, and was not replaced.
The algal biomass in the samples was determined by fluorescence measurement (BIO-TEK Multi-Detection Microplate Reader, Model FLx800). The measurements were performed at least in duplicate.
The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and biomass and their 95% confidence intervals were calculated by Probit Analysis.
For the determination of the LOEC and NOEC, average growth rate and biomass at the test concentrations were compared to the control values by Williams t-test.
- Other: At the end of the test, a sample was taken from the control and from the test concentration of nominal 12 mg/L to determine a potential influence of the test item on the algal cells. The shape and size of the algal cells were visually inspected.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study
- Test concentrations: 0, 1.0, 10 and 100 mg/L in the first pre-test (pH 7.8 - 8.5); 0, 0.01, 0.1, 1.0 and 10 mg/L in the second pre-test (pH 8.1 - 8.4); 0, 0.01, 0.10, 1.0 and 10 mg/L in the third pre-test (pH 6.5 - 6.6)
- Results used to determine the conditions for the definitive study: The test concentrations were based on results of a range-finding test and of a pre-experiment to determine water solubility of the test item (non-GLP).

Reference substance (positive control):

yes

Remarks:

(potassium dichromate)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.29 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 1.96 - 2.60 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

3.82 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 3.45 - 4.16 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10.17 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 9.44 - 11.07 mg/L

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.46 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The test item had a statistically significant inhibitory effect on the growth of the algae (average growth rate and biomass) after the test period of 72 hours at the mean measured concentration of 0.46 mg/L and at all higher test concentrations (results of Williams t-tests, one-sided, alpha = 0.05). Thus, this concentration was determined to be the 72 hour LOEC.

The 72-hour NOEC was determined to be 0.20 mg/L, since up to and including this mean measured test concentration, the growth rate and biomass of the algae after 72 hours were not statistically significantly lower than in the control.

The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 12 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.

No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

Results with reference substance (positive control):

The result of the latest positive control test performed in March 2010 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 0.94 mg/L (study C86922), range of the 72-hour EC50 for the growth rate from 2000 to 2010: 0.71 1.7 mg/L).

Any other information on results incl. tables

The measured concentrations of 4-TBC in the test media of the nominal test concentrations of 0.25 to 12 mg/L were between:

- 91 and 100% of the nominal values at the start of the test

- 86 and 96% of the nominal values after 24 hours test duration

- 74 and 85% of the nominal values after 48 hours test duration

- 64 and 81% of the nominal values at the end of the test

During the test period of 72 hours, a decrease of test item concentration in the test media occurred. The mean measured test concentrations (calculated as the geometric means of the concentrations measured at the start and the end of the test) were between 78 and 90% of the nominal values (see table 3).

Additionally, the media of the nominal concentrations of 0.55 and 5.55 mg/L were incubated over the test duration under the same test conditions as well as in the dark but without algae. The mean measured concentrations (calcultated as geometric means) of these test item samples were in the range of 83 to 92% of nominal. The comparison between light and dark incubation did not result in a distinct difference in the concentrations (see table 4).

Table 1: Average Growth Rates (µ)

Nominal test item concentration	Mean measured concentration	Average growth rate µ (day^-1) and inhibition of µ (Ir)
		0-24 h		0-48 h		0-72 h
(mg/L)	(mg/L)	µ	Ir(%)	µ	Ir(%)	µ	Ir(%)
Control	---	1.68	0.0	1.76	0.0	1.61	0.0
0.25	0.20	1.63*	3.1	1.76	0.0	1.60	0.4
0.55	0.46	1.55*	8.0	1.70*	3.4	1.58*	1.8
1.20	0.99	1.58*	6.1	1.70*	3.6	1.58*	1.7
2.55	2.10	1.65*	2.1	1.63*	7.5	1.52*	5.4
5.55	4.73	1.66*	1.1	1.31*	25.6	1.14*	28.9
12.0	10.8	1.54*	8.7	1.04*	40.9	0.79*	50.7

 *: mean value significantly lower than in the control (according to Williams t-test, one-sided, alpha = 0.05)

Table 2: Biomass (Y)

Nominal test item concentration	Mean measured concentration	Biomass Y (x 10^3) and inhibition of Y (Iy)
		0-24 h		0-48 h		0-72 h
(mg/L)	(mg/L)	Y	Iy(%)	Y	Iy(%)	Y	Iy(%)
Control	---	6.9	0.0	52.1	0.0	194.9	0.0
0.25	0.20	6.5*	6.2	52.1	0.0	190.8	2.1
0.55	0.46	5.9*	15.4	46.0*	11.6	179.0*	8.2
1.20	0.99	6.1*	12.0	45.7*	12.3	179.6*	7.8
2.55	2.10	6.6*	4.2	39.7*	23.9	150.1*	23.0
5.55	4.73	6.8*	2.3	20.2*	61.3	47.1*	75.8
12.0	10.8	5.8*	16.6	11.1*	78.6	15.5*	92.1

*: mean value significantly lower than in the control (according to Williams t-test, one-sided, alpha = 0.05)

Table 3: Mean measured test item concentration

Nominal test item concentration	Mean measured concentration of the test item (geometric mean)
[mg/L]	[mg/L]	[% of nominal]
0.25	0.20	78
0.55	0.46	84
1.20	0.99	83
2.55	2.10	82
5.55	4.73	85
12.0	10.8	90

Table 4: Mean measured test item concentration: illuminated and dark

Nominal test item concentration	Measured test item concentration
	algae + illuminated		illuminated		dark
[mg/L]	[mg/L]	[% of nominal]	[mg/L]	[% of nominal]	[mg/L]	[% of nominal]
0.55	0.460	84	0.455	83	0.509	92
5.55	4.73	85	4.95	89	4.76	86

Table 5: First pre-test: Effects of 4 -TBC on Pseudokirchneriella subcapitata, pH 7.8 - 8.5, exposure period: 72h

Nominal test item concentration (mg/L)	% biomass inhibition to control
Control	--
1.0	73
10	98
100	99

Table 6: Second and third pre-tests: Effects of 4 -TBC on Pseudokirchneriella subcapitata, exposure period: 72h

Nominal test item concentration mg/L	2^ndPre-test, pH 8.1 – 8.4	3^rdPre-test, pH 6.5 – 6.6
	% biomass inhibition to control	% biomass inhibition to control
Control	--	--
0.010	8.0	-4.5
0.10	11	6.0
1.0	63	5.9
10	93	85

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

(biomass parameter factor in the controls > 16, mean coefficient of variation of the daily growth rates in the control < 35%, coefficient of variation of mean specific growth rate in the controls < 7%)

Conclusions:

Based on the results of this study, 4-tert-butylpyrocatechol would be considered as harmful for algae.

Executive summary:

The influence of 4 -tert-butylpyrocatechol (4 -TBC) on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72 -hour static test (Harlan, 2010) according to the Commission Regulation (EC) n° 440/2008, Part C.3 and the OECD Guideline for Testing of Chemicals, n°201 (2006).

The nominal concentrations of the test item of 0.25, 0.55, 1.20, 2.55, 5.55 and 12 mg/L were tested in parallel with a control.

The measured concentrations of 4 -TBC in the test media of the nominal test concentrations of 0.25 to 12 mg/L were between 91 and 100% of the nominal values at the start of the test and 64 and 81% of the nominal values at the end of the test. During the test period of 72 hours, a decrease of test item concentration in the test media occurred.

The biological results were to related to the mean measured test item concentrations (calculated as the geometric means of the concentrations measured at the start and the end of the test):

Nominal test item concentration	Mean measured concentration of the test item (geometric mean)
[mg/L]	[mg/L]	[% of nominal]
0.25	0.20	78
0.55	0.46	84
1.20	0.99	83
2.55	2.10	82
5.55	4.73	85
12.0	10.8	90

Additionally, the media of the nominal concentrations of 0.55 and 5.55 mg/L were incubated over the test duration under the same test conditions as well as in the dark but without algae. The mean measured concentrations (calculated as geometric means) of these test item samples were in the range of 83 to 92% of nominal. The comparison between light and dark incubation did not result in a distinct difference in the concentrations (see table below).

Nominal test item concentration	Measured test item concentration
	algae + illuminated		illuminated		dark
[mg/L]	[mg/L]	[% of nominal]	[mg/L]	[% of nominal]	[mg/L]	[% of nominal]
0.55	0.460	84	0.455	83	0.509	92
5.55	4.73	85	4.95	89	4.76	86

The test item had a statistically significant inhibitory effect on growth of the algae (average growth rate and biomass) after the test period of 72 hours at the mean measured concentration of 0.46 mg/L and at all higher test concentrations (results of Williams t-tests, one-sided, alpha = 0.05). Thus, this concentration was determined to be the 72 -hour LOEC.

The 72 -hour NOEC was determined to be 0.20 mg/L, since up to and including this mean measured test concentration, the growth and biomass of the algae after 72 hours were not statistically significantly lower than in the control.

The biological results were summarized as follows:

Parameter	Growth rate	Biomass
(0-72 h)
EC10  (mg/L)	2.29	1.37
95% confidence interval	1.96 – 2.60	1.07 – 1.62
EC20  (mg/L)	3.82	1.83
95% confidence interval	3.45 – 4.16	1.52 – 2.09
EC50  (mg/L)	10.17	3.18
95% confidence interval	9.44 – 11.07	2.87 – 3.53
NOEC (mg/L)	0.20	0.20
LOEC (mg/L)	0.46	0.46

Based on the results, 4 -TBC was harmful for algae.

This study is classified as acceptable and satisfies the guideline requirements for acute algae toxicity study.