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Endpoint:
toxicity to microorganisms, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: peer-reviewed technical publication. method based on OECD method for COD. Acclimation period included in standard method.
Reason / purpose for cross-reference:
reference to other study
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD, 1971: Pollution by detergents. Determination of the biodegradability of anionic sunthetic surface active agents
Deviations:
not specified
Qualifier:
equivalent or similar to guideline
Guideline:
other: Verordnung uber die abbaubarkait von detergentien in wash- und reinigungslittlen, 1 sept 1962
Deviations:
not specified
Principles of method if other than guideline:
the two tests provide a suitable standard model of an activated sludge treatment plant.
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): perborate
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc):
Test organisms (species):
activated sludge
Details on inoculum:
- Preparation of inoculum for exposure: Activated sludge is devided equally between two plants. 2 plants were operated as per specification and half of the sludge or volume of waste water was exchanged daily between the two tanks until both showed > 80% BMAS reduction and a satisfactory sludge density (2g/l TM).
- Pretreatment: plants are loaded with 125 or 250 mg/l of boiled perborate equivalent to 10 respectively 20 mg/l boron. The plant is then operated for two weeks before starting the two weeks evaluation period.
Test type:
not specified
Water media type:
not specified
Limit test:
no
Total exposure duration:
3 h
pH:
7.4 (7.10-7.80)
Nominal and measured concentrations:
125 mg/L, 250 mg/L, 500 mg/L perborate
Details on test conditions:
SAMPLES
- 2 synchronously working plants:
* one fed with synthetic waste water to which 20 mg/l of sodium dodecyl benzene sulphonate was added.
* one loaded with increasing concentrations of boiled perborate.
Reference substance (positive control):
not specified
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: treatment plant performance
Remarks on result:
other: usable for PNEC-stp derivation
Reported statistics and error estimates:
each series of measurements was studied by Kolmogorov-Smirnov for standard distribution.

LOEC: 120 mg B/L

Validity criteria fulfilled:
yes
Remarks:
earlier version of OECD activated sludge was used and followed
Conclusions:
This study is a peer-reviewed technical publication. The study method is based on an OECD method for COD. The acclimation period is included in the standard method.
Endpoint:
toxicity to microorganisms, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Unpublished Henkel KGaA
Principles of method if other than guideline:
Not stated
GLP compliance:
not specified
Test organisms (species):
Entosiphon sulcatum
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
18 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to GLP and international guidelines
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Principles of method if other than guideline:
The study was conducted according to the Commission Directive 88/302/EEC, official journal of the EC L 133 Part C (1998). This test method is in most parts identical with OECD 209.
GLP compliance:
yes (incl. QA statement)
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Type: Mixed population of aquatic microroganisms
- Origin: aeration tank of a waste water treatment plant treating predominantly domestic sewage (Wupper area water authority)
- Pretreatment: aeration of the activated sludge, daily feed with synthetic medium
- incubation time: 3h with permanent aeration
Water media type:
freshwater
Test temperature:
20 +/- 2 °C
pH:
7.9
Dissolved oxygen:
5.6 - 5.8
Nominal and measured concentrations:
- 100, 1000 and 10000 mg/L
Details on test conditions:
- A physico chemical oxygen consumption control is carried out since some substances can also consume oxygen by chemical reactivity. in order to be able to tell the difference between physicochemical oxygen consumption and biological oxygen consumption (respiration), at least the maximum concentration of the test substance is tested without activated sludge.
- Type of application: direct weighing
- test concentration of the activated sludge: 400 mg/L suspended solids
Reference substance (positive control):
yes
Remarks:
3.5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- Bor amorph Grade I 95/97% showed 3.3 % respiration inhibition of activated sludge at a test substance concentration of 10,000 mg/L.
Results with reference substance (positive control):
EC50 = 11 mg/L (determined by probit analysis)
Validity criteria fulfilled:
yes
Remarks:
respiratory rate of the 2 controls differs less than 15 %, respiratory rate of the controls <60 mg O2/l.h, EC50 of the reference substance is in the range 5-30 mg/L
Conclusions:
Bor amorph grade I 95/97% showed 3.3 % respiration inhibition of activated sludge at a test substance concentration of 10,000 mg/L.
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Peer-reviewed technical publication. Data not presented.
Principles of method if other than guideline:
Test organisms were exposed during 72h in petri dishes
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
no
Test organisms (species):
Entosiphon sulcatum
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Nominal and measured concentrations:
Nominal concentrations : 10, 15, 20, 50 and 110 mg B/l
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
15 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
growth inhibition
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
growth inhibition
Endpoint:
toxicity to microorganisms, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Peer-reviewed technical publication. Data not presented.
Principles of method if other than guideline:
Test organisms were exposed during 72h in petri dishes
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
no
Test organisms (species):
other: Opercularia bimarginata
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Nominal and measured concentrations:
Nominal concentrations : 10, 15, 20, 50 and 110 mg B/l
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
growth inhibition
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
growth inhibition
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Relevant study for activated sludge
Principles of method if other than guideline:
Warburg oxygen uptake measurements were conducted to evaluate the effect boron would have on the respiration rate of a municipal activated sludge.
GLP compliance:
not specified
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Origin : Marlborough Westerly Municipal Treatment Plant in Massachusetts.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 h
Test temperature:
20°C
Nominal and measured concentrations:
Nominal concentrations : 0, 10, 20, 50, 100, 200 and 300 mg/l
Details on test conditions:
TEST MEDIUM / WATER PARAMETERS
-The activated sludge had a suspended solids level of 3400 mg/l.
Duration:
7 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Endpoint:
activated sludge nitrification inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Reliable study but not according to international guidelines
Principles of method if other than guideline:
The effect of boric acid in wastewater on nitrification was investigated in a continuous nitrification process in an aerobic upflow fixed bed reactor.
GLP compliance:
not specified
Test organisms (species):
other: Fixed bed reactor with nitrifying organisms.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Remarks on exposure duration:
Duration not relevant, could take several weeks before addaptation.
Test temperature:
20-22 °C
pH:
7,4 - 7,5
Nominal and measured concentrations:
Nominal concentrations ranging from 128 - 1026 mg B/l
Details on test conditions:
The effect of boric acid in wastewater (128–1026 mg B/l) on nitrification was investigated at T = 20 °C, pH = 7,4 and at substrate concentrations of up to 70 mg/l NH4-N and NO2-N in batch experiments in the “Karlsruher Flasche”. In a continuous nitrification process in an aerobic upflow fixed bed reactor at T = 22 °C and pH = 7,5, 2 g NH4-N/m2/d were fed at a hydraulic retention time of 3 days.
Dose descriptor:
NOEC
Effect conc.:
500 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
inhibition of nitrification rate
Remarks on result:
other: Test duration not clear, but it could take some days for adaptation.
Details on results:
Boric acid inhibited nitrite formation, whereas the oxidation of nitrite to nitrate was stimulated. In the continuously run fixed bed reactor a nitrification efficiency of 99,5 % was achieved up to boric acid concentrations of 500 mg B/l. At a starting concentration of 200 mg/l NH4-N the NH4-N concentrations in the effluent were less than 1,0 mg/l. Approximately 0,2 mg/l NO2-N were found in the presence of boric acid, whereas more nitrite (1,0 mg/l NO2-N) was found in absence of boric acid. When 600 mg/l B were sup- plied, nitrification activity was reduced to 55%. The reactor recovered to 98% nitrification efficiency after 5–7 weeks, the total adaptation time for achieving complete nitrification at 700 mg/l B was more than 28 weeks. At boric acid concentrations higher than 700 mg/l B no stable nitrification could be achieved.
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Principles of method if other than guideline:
TNO sample code of the reference substance was 97-0034-C and not 91-0002-B as stated in the protocol
The batch no. of the reference substance was D7, 060-0 and not 00410-055.
These deviations are assumed not to have affected the results of the study.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Water solubility (under test conditions): 4.7% at 20°C
- Purity: 99.9%
Analytical monitoring:
not specified
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- a stock solution of the test substance was prepared by dissolving 1006 g of boric acid, manufacturing grade, in 500 ml of BOD dilution water
- The BOD dilution water was prepared according to NEN Standard N° 6634
- The synthetic sewage feed was prepared according to OECD guideline N° 209
Test organisms (species):
activated sludge
Details on inoculum:
- Culture: 20l of activated sludge was taken from an oxidation ditch situated at the district of Hazerswoude Dorp, The Netherlands.
- Preparation of inoculum: sample was centrifuged and the supernatant discarded. Activated sludge was washed three times by centrifugation (10 min at 2000 rpm = max 1218g) and resuspension in tap water twice and once in dilution water. Suspension was diluted to 4.9 g/l of MLSS by addition of dilution water aerated vigorously and kept at aprox 20°C in the dark. 50ml of synthetic sewage feed was added per litre of sludge suspension before the sludge was used the following day.
- Pretreatment: The dry weight was checked before commencement of the test and readjusted to a mixed liquor suspended solids level of 3.7 g/l
Test type:
not specified
Water media type:
not specified
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 +/- 2°C
pH:
7.5
Nominal and measured concentrations:
nominal concentrations: 0, 3.2, 10, 32, 100, 320 and 1001 mg/l.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: BOD bottle, volume 295 ml
- Aeration: vigorously during 10 minutes


INCUBATION
- Incubation of the samples was tarted at intervals of 15 minutes and the first and last sample were controls without the test substance.
- The suspensions were aerated vigorously and incubated at approx 20°C for exactly 3 hours.
- After 3h of incubation, a sample of each mixture was poured into a BOD bottle and stirred vigorously on a magnetic stirrer.
- An oxygen electrode was inserted into the sample and the oxygen concentration in the sample was continuously measured.
- The oxygen concentration was recorded every minute during a period of about 10 minutes.


MIXTURES PREPARING
- control mixture of 16ml synthetic sewage feed and 284 ml dilution water were prepared in one litre glass beakers.
- text mixtures were prepared by combining 16 ml synthetic sewage feed with the appropriate volumes of the test substance stock solutions and dilution water to a volume of 300 ml. the test was started by adding 200 ml of sludge suspension to the mixtures with and without test substance.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 175 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: usable for PNEC-stp derivation. However this is unbounded value based on NOEC >=17.5 mg B/L
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
112 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% confidence interval of 495-821 mg/l
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
35.4 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: preferred for PNEC-stp derivation. EC10 value calculated from original data
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
17.5 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: Value used for PNEC in RAR
Results with reference substance (positive control):
- Results with reference substance valid? yes, within the range prescribed by OECD guideline 209
- Relevant effect levels: EC50 of 3,5-dichlorophenol was 11,5 mg/l with a 95% confidence interval of 6,3 - 21 mg/l

3 h NOEC: 17.5 mg/L, but calculated without any statistical method.

Validity criteria fulfilled:
yes
Remarks:
complies with GLP practice
Conclusions:
This study follows the OECD 209 guidelines and is well documented. Standard test methods reffered to in the relevant directive. No deviations from the test guideline have been detected. The study is rated as highly reliable in quality and is consistent with earlier tests of activated sludge. This is the preferred test for derivation of the PNEC stp.
Endpoint:
toxicity to microorganisms, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Peer-reviewed technical publication. Data not presented.
Principles of method if other than guideline:
Test organisms were exposed during 72h in petri dishes
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
no
Test organisms (species):
Paramaecium caudatum
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Nominal and measured concentrations:
Nominal concentrations : 10, 15, 20, 50 and 110 mg B/l
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
growth inhibition
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
growth inhibition

Description of key information

Taking into account the extensive information on the toxicity of boron to microorganism in STP,

i. e. - 4 respiration studies using activated sludge, showing NOEC between 17.5 and >10000 mg B/L, - 1 nitrification study in a fixed bed reactor, showing no effect up to 500 mg B/L, and - 3 studies of growth of protozoans relevant for the functioning of STPs, show NOEC data between 10 and 20 mg B/L. Therefore, it was decided to base the PNECstp on the lowest NOEC from the available toxicity data. The lowest NOEC for the protozoan Opercularia bimarginata on growth is 10 mg B/L (Guhl. 2000). Since the studies of STP functioning showed no adverse effects at higher concentrations, no additional assessment factor was used, therefore resulting in e PNECstp of 10 mg B/L.

Please refer to read-across statement section 13.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
10 mg/L

Additional information

7.4.1 Respiration Inhibition

Four tests of sludge respiration are available. Hanstveit and Schoonmade (2002) tested the effect of boric acid on sludge respiration according to the OECD 209 guidelines. The purpose of this guideline is to provide a rapid screening method, which is not designed to derive EC10 values. Hanstveit and Schoonmade reported an EC20 of 112 mg B/L (95% CI 87 to 144 mg B/L), and an unbounded EC50 which exceed the maximum exposure of 175 mg B/L. Actual inhibition at the maximum exposure was 24%. They reported a NOEC of 17.5 mg B/L and LOEC of 56 mg B/L, although this may not be based on a statistical measure as the systems were not replicated. Muller and Burns (2001) tested amorphous boron and found a NOEC of >10000 mg/L.

Gerike et al. (1976) evaluated methylene blue activity reductions (MBAS test) and COD reduction (OECD, 1971) and reported that addition of 20 mg B/L had no adverse effects on a model STP. In fact, MBAS and COD removal was enhanced by boron relative to controls. No higher boron concentrations were tested. The actual substance tested was a sodium perborate which had been boiled to remove oxygen.

Webber et al. (1977) conducted Warburg oxygen uptake measurements to evaluate the effect boron would have on the respiration rate of a municipal activated sludge. They found a NOEC of 100 mg B/L. In later work, they observed no difference in COD removal in laboratory-scale activated sludge plants operating with 0, 100 or 300 mg B/L.

 

7.4.2 Nitrification efficiency

Effects of boron on nitrifying microorganisms are reported by Buchheister & Winter (2003). This study shows that in a continuously run fixed bed reactor a nitrification efficiency of 99,5% was achieved up to boric acid concentrations of 500 mg B/l. At a starting concentration of 200 mg/L NH4-N the NH4-N concentrations in the effluent were less than 1,0mg/l. However, when 600 mg/L B was supplied, nitrification activity was reduced to 55%.

 

7.4.3 Micro-organism toxicity

Toxicity data on protozoans (ciliates and flagellates) which are relevant for STP show NOECs between 10 and 20 mg/L (Guhl 2000). The protozoans for which toxicity data are available on the growth inhibition are Entosiphon sulcatum, Paramecium caudatum and Opercularia bimarginata. These protozoans have been found in activated sludge from sewage treatment plants in Brazil, France, Germany and Spain (Amann et al., 1998; Motta et al., 1998 and Pérez-Uz et al., 2010).

 

7.4.4 STP data

Monitoring data from studies on a pilot-scale plant indicate that once microorganisms have been adapted to the presence of boron they can tolerate boron at a concentration of at least 3 mg B/L (Umweltbundesamt BE121, 2000). The measured arithmetic average influent concentrations were 3.0 mg B/L (min. 1.6; max. 4.9 mg B/L) in unfiltered samples collected after primary treatment. The function (nitrogen and carbon - removal) of the pilot-scale plant was not affected. Unfortunately, boron data for full-scale plants which include good performance data are missing. Zessner et al. (2003) monitored average boron concentrations in the effluent of two Austrian full-scale STPs. The average concentration was 1.31 mg B/L for STP1 and 0.78 mg B/L for STP2. ECETOC (1997) reported influent and effluent concentrations between 0.27-0.78 and 0.39 -0.75 mg B/L. These studies do not permit conclusions about higher boron concentrations, but suggest that, under current use conditions, boron concentrations are below those seen for all the laboratory studies described above.

The following information is taken into account for effects on aquatic micro-organisms for the derivation of PNEC:

Taking into account the extensive information on the toxicity of boron to microorganism in STP, i. e.

- 4 respiration studies using activated sludge, showing NOEC between 17.5 and >10000 mg B/L,

- 1 nitrification study in a fixed bed reactor, showing no effect up to 500 mg B/L, and

- 3 studies of growth of protozoans relevant for the functioning of STPs, show NOEC data between 10 and 20 mg B/L.

Therefore, it was decided to base the PNECstp on the lowest NOEC from the available toxicity data. The lowest NOEC for the protozoan Opercularia bimarginata on growth is 10 mg B/L (Guhl, 2000). Since the studies of STP functioning showed no adverse effects at higher concentrations, no additional assessment factor was used, therefore resulting in a PNECstp of 10 mg B/L.