1,4-dimethoxybenzene

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 27 apr 2007 to 30 may 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the EC and OECD guidelines and GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae), and at the end of the test (containing algae). At the same sampling times, duplicate samples were also taken from the control. For sampling at the end of the test, the test medium of the treatment replicates was pooled.
- Sampling method: no data
- Sample storage conditions before analysis: All samples were deep-frozen immediately after sampling and protected from light until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low water solubility of the test item, a supersaturated dispersion of the test item with a loading rate of 100 mg/L was prepared
by dispersing 59.0 mg of the test item into 590 mL of test water using ultrasonic treatment for 15 minutes. No auxiliary solvent or emulsifier was
used. This supersaturated dispersion was stirred by a magnetic stirrer at a room temperature in the dark over 3 hours to dissolve or disperse a
maximum amount of the test item. The stirring period of 3 hours was chosen according to the results of a pre-test (non-GLP) which showed that the solution equilibrium was reached after this time. The pre-test was performed in the dark using test media without addition of algae. The stability of the test item over at least four days was proven. The vessel was sealed tightly to avoid losses by volatization.

The supersaturated dispersion of the test item with the maximum loading rate of 100 mg/L was filtered through a membrane filter at reduced
pressure (Schleicher & Schuell, Type NC45, pore size 0.45 µm) after the 3 hours stirring period just before preparation of the test media. This
undiluted filtrate served as the medium with the highest test concentration. It was used to prepare the lower test concentrations, as well, by diluting
aliquots with test water. The test media were prepared just before the start of the test (= addition of algae).

- Differential loading: no
- Controls: yes, test water without test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Clear test media were observed throughout the test.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchnerella subcapitata
- Strain: N° 61.81 SAG
- Source (laboratory, culture collection): supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen,
D-37073 Göttingen, Germany).
- Age of inoculum (at test initiation): the cells at the start of the test were taken from an exponentially growing pre-culture, which was set up four days prior to the test under the same conditions as in the test. One day before the start of the test, the pre-culture was diluted threefold to keep the algae in exponential growth.
- Method of cultivation: The algae are cultivated in RCC’s laboratories under standardized conditions according to the test guidelines.

ACCLIMATION: none

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no

Hardness:

24 mg/L as CaCO3 (as calculated)

Test temperature:

22-23°C

pH:

8.1 to 9.2

Dissolved oxygen:

no data

Salinity:

no data

Nominal and measured concentrations:

Nominal: undiluted supersaturated and filtrated solution (100 mg/l), and dilutions to 1/3.2; 1/10; 1/32; 1/100 and 1/320
Measured at the start: undiluted gave 87.4 mg/l, 1:32 gave 2.68 mg/l; 1:10 gave 8.64 mg/l; 1:3.2 gave 26.9 mg/l
Measured at the end: undiluted gave 49.7 mg/l, 1:32 gave 1.42 mg/l; 1:10 gave 5.36 mg/l; 1:3.2 gave 17.3 mg/l
Geometric mean measured : undiluted gave 65.9 mg/l, 1:32 gave 1.95 mg/l; 1:10 gave 6.8 mg/l; 1:3.2 gave 21.6 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks filled with 15 ml algal suspension
- Type (delete if not applicable): closed: the flasks were covered with glass dishes
- Aeration: no, but stirred continuously by magnetic stirrers
- Renewal rate of test solution (frequency/flow rate): no
- Initial cells density: 10000 cells/ml
- Control end cells density: Control: mean 130.6 (*10000 cells/mL) (SD:8.3; n=6) at 72h
- No. of vessels per concentration (replicates):3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): none

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
The algae were cultivated and tested in synthetic test water, prepared according to the test guidelines. Analytical grade salts were dissolved in sterile purified water to obtain the following final nominal concentrations:
Macro-nutrients:
NaHCO3 50.0 mg/L
CaCl2 , 2 H2O 18.0 mg/L
NH4Cl 15.0 mg/L
MgSO4 , 7 H2O 15.0 mg/L
MgCl2 , 6 H2O 12.0 mg/L
KH2PO4 1.6 mg/L
Trace elements:
Na2EDTA , 2 H2O 100.0 µg/L
FeCl3 , 6 H2O 80.0 µg/L
MnCl2 , 4 H2O 415.0 µg/L
H3BO3 185.0 µg/L
Na2MoO4 , 2 H2O 7.0 µg/L
ZnCl2 3.0 µg/L
CoCl2 , 6 H2O 1.5 µg/L
CuCl2 , 2 H2O 0.01 µg/L

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L as CaCO3).

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: no
- Light intensity and quality: and irradiated continuously at a measured light intensity of about 7200 Lux (mean value), range: 6270 to 7980 Lux (minimum and maximum value of measurements at nine places distributed over the experimental area at the surface of the test media). This irradiation was achieved by fluorescent tubes (Philips TLD 36W-1/840), installed about 35 cm above the test flasks.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The algal cell densities in the samples were determined by counting with an electronic particle counter (Coulter Counter, Model ZM), with at least two measurements per sample.
- Chlorophyll measurement: no
- Other: in addition, after 72 hours exposure, a sample was taken from the control and from a test concentration with reduced algal growth (undiluted filtrate) and the shape and size of the algal cells were examined microscopically.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2. The test concentrations were based on the results of a range-finding test (non-GLP). This enlarged spacing factor of 3.2 was chosen since the results of the range-finding test indicated that a large concentration range was necessary for the definitive test to determine the dose-response relationship
- Justification for using less concentrations than requested by guideline: not applicable, since 5 concentrations tested, as required.
- Range finding study
- Test concentrations and Results used to determine the conditions for the definitive study: not reported, since rang finding study was not performed in GLP.
- :

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

50.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 39.4-60.5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

23.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 10.5-32.4

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

30.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 17-39.3

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

> 65.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: not determined

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: no data

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

21.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: no data

Details on results:

- Exponential growth in the control (for algal test): yes, the biomass increased by a factor of 177 over 72 h. The validity criterion of increase of cell density by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates (section-by-section growth rates) was 14.4% after 72 hours and, thus, lower than the maximum of 35% given by the OECD test guideline. The coefficient of variation of the average specific growth rates in the control replicates during the test period of 72 hours was 1.3% which is below the maximum of 7% indicated by the OECD test guideline. Therefore, all validity criteria given by the test guidelines were fulfilled.
- Observation of abnormalities (for algal test): The microscopic examination of the algal cells after 72 hours exposure showed no difference between the algae growing in the undiluted filtrate and the algal cells in the control. The shape and size of the algal cells growing in test media containing the test item at and up to this test concentration were not affected.

At the start of the test, the measured test item concentrations in the analyzed test media samples (dilutions 1:32, 1:10,1:3.2 and the undiluted
filtrate) amounted to 2.68, 8.64, 26.9 and 87.4 mg/L . After 72 hours, the measured concentrations of Paradimethoxybenzene in these test media
decreased to 1.42, 5.36, 17.3 and 49.7 mg/L. This decrease of the test item concentrations may possibly have been caused by a photolytic
degradation or by uptake of the algal cells.
The test item had a statistically significant inhibitory effect on the growth (yield) of Pseudokirchneriella subcapitata after the test period of 72 hours
at the test concentrations of 6.8 mg/L and above (results of Dunnett’s test, one-sided, a = 0.05). The growth rate µ was statistically significantly reduced at the next higher test concentration of 21.6 mg/L .

Results with reference substance (positive control):

For evaluation of the algal quality and the experimental conditions, potassium dichromate is tested as a positive control at least once a year to
demonstrate satisfactory conditions of the test. The latest result of the positive control test in 2006 (72-h EC50 for the growth rate: 1.70 mg/L, RCC Study No. B13151) showed that the toxic performance was valid and within the historical range of the RCC laboratory (from 2000 to 2006: 72-h
EC50: 0.71–1.74 mg/L).

Reported statistics and error estimates:

ErC50 and EyC50 values, and the corresponding EC10 value and EC90 values and their 95% confidence limits were calculated as far as possible by Probit Analysis.
For the determination of the LOEC and NOEC, the calculated mean yield and the average growth rate at the test concentrations were tested for significant differences when compared to the control values by Dunnett’s tests.

Growth rates µ and percentage inhibition of µ (I_r) during the test period:

Treatment / dilution	Mean measured concentration	Growth rate µ and % inhibition of µ
		0-24 h		0-48 h		0-72 h
	(mg/L)	µ (1/day)	Ir (%)	µ (1/day)	Ir (%)	µ (1/day)	Ir (%)
Control	/	1.872	0.0	1.718	0.0	1.623	0.0
1:320	n.a.	1.939	-3.6	1.728	-0.6	1.594	1.8
1:100	n.a.	1.906	-1.8	1.700	1.0	1.561	3.9
1:32	1.95	1.894	-1.2	1.723	-0.3	1.592	1.9
1:10	6.80	1.896	-1.2	1.731	-0.8	1.569	3.4
1:3.2	21.6	1.701 *	9.1	1.632 *	5.0	1.501 *	7.6
Undiluted filtrate	65.9	0.993 *	47.0	0.501 *	70.8	0.529 *	67.4

- % inhibition: increase in growth relative to that of control

n.a.: not analyzed

*:     mean value significantly lower than in control (according to a Dunnett’s test, one-sided smaller, a = 0.05)

Validity criteria fulfilled:

yes

Conclusions:

Paradimethoxybenzene is harmful for algae (growth rate and yield).

Executive summary:

The influence of the test item Paradimethoxybenzene on the growth of the freshwater green algal species Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) was investigated in a 72-hour static test according to the EU Commission Directive 92/69/EEC, C.3 (1992), and the OECD Guideline 201 (2006).

Due to the low water solubility of the test item, a supersaturated dispersion of the test item with a loading rate of 100 mg/L was continuously stirred at a room temperature in the dark over 3 hours. Then, the stock dispersion was filtered. The undiluted filtrate of the stock dispersion and the dilutions 1:3.2, 1:10, 1:32, 1:100 and 1:320 were used as test media. Additionally, a control was tested in parallel.

At the start of the test, the measured test item concentrations in the analyzed test media samples (dilutions 1:32, 1:10, 1:3.2 and the undiluted filtrate) amounted to 2.68, 8.64, 26.9 and 87.4 mg/L. After 72 hours, the measured concentrations of Paradimethoxybenzene in these test media, incubated during the test period under the test conditions with algae decreased to 1.42, 5.36, 17.3 and 49.7 mg/L.

All biological results are based on the mean measured test item concentrations of 1.95 mg/L (dilution 1:32), 6.80 mg/L (dilution 1:10), 21.6 mg/L (dilution 1:3.2) and 65.9 mg/L (undiluted filtrate), calculated as the geometric mean over all measurements per test concentration during the test period.

The 72-hour NOEC and LOEC for yield were determined to be 1.95 and 6.8, respectively. The 72-hour NOEC and LOEC for growth rate were determined to be 6.8 and 21.6 mg/L, respectively. The EC50 for yield and growth rate were determined to be 24.9 and 50.5 mg/L, respectively.

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

Description of key information

The influence of the test item Paradimethoxybenzene on the growth of the freshwater green algal species Pseudokirchneriella subcapitatawas investigated in a 72H static test according to OECD Guideline 201 and GLP. The EC50 for yield and growth rate were determined to be 24.9 and 50.5 mg/L, respectively (based on the mean measured test item concentrations, calculated as the geometric mean over all measurements per test concentration during the test period). The 72-hour NOEC and LOEC for yield were determined to be 1.95 and 6.8, respectively. The 72-hour NOEC and LOEC for growth rate were determined to be 6.8 and 21.6 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

50.5 mg/L

EC10 or NOEC for freshwater algae:

6.8 mg/L

Additional information

Three studies were available, but only one (RCC, 2006) was of reliability 1 according to Klimisch and was selected as key study.

The two other studies were of reliability 3, they were poorly described, so were not taken into account for the assessment.

In the key study, (RCC, 2006),