Carbon black

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

distribution

Qualifier:

no guideline followed

GLP compliance:

not specified

Specific details on test material used for the study:

Carbon black (Association of Powder Process Industry and Engineering, Kyoto, Japan).
Fraction of CB particles below 100 nm was approximately 3% and 40% of the nominal concentrations of CB particles by weight and number.

Species:

rat

Strain:

Sprague-Dawley

Route of administration:

inhalation

Details on exposure:

Median particle diameter 116.4 nm

Duration and frequency of treatment / exposure:

6 hours per day, 5 days per week for a total of 4 weeks; mean CB particle size (nm) ± SD determined at 1, 8, 15, 22 and 29 days after exposure was 118.1 ± 2.4, 119.1 ± 2.7, 122.2 ± 2.0, 122.4 ± 2.5, and
121.9 ± 3.6, respectively. Concentration of CB particles below 100 nm was approximately 3% and 40% of the nominal concentrations of CB particles by weight and by number, respectively

Dose / conc.:

15.6 mg/m³ air

Control animals:

yes

Details on study design:

Rats were exposed to nominal concentrations of 15.6±3.5 mg/m3 (1.57±0.4×1010 particle number/m3) for 6 h/day, 5 days per week for a total of 4 weeks. The control rats were exposed to clean, filtered air containing no CB for the same period. The concentrations of CB were monitored twice weekly by measuring the gross weight with a PTFE binder glass filter
Mean size (nm)±SD determined at 1, 8, 15, 22 and 29 days after exposure was 118.1±2.4, 119.1±2.7, 122.2±2.0, 122.4±2.5, and 121.9±3.6, respectively. Concentration of CB particles below 100 nm was approximately 3% and 40% of the nominal concentrations of CB particles by weight and number

Type:

absorption

Results:

no evidence of systemic absorption

Type:

distribution

Results:

no evidence of translocation of inhaled carbon black

Type:

metabolism

Results:

not measured

Executive summary:

Evidence of translocation of inhaled carbon black (median diameter of 116 nm) was not obtained. It is likely that inhaled nano-sized carbon black particles form aggregations in the lung and do not exert direct adverse effects on extrapulmonary tissues.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

calculation (if not (Q)SAR)

Remarks:

MPPD Modelling

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

exposure-related information

Objective of study:

toxicokinetics

other: deposition kinetics in lungs

Principles of method if other than guideline:

Dustiness of the test substance was investigated in accordance with rotating drum method and the results expressed in mg/g. The fraction of material that becomes airborne under the conditions of this test is described as “total dustiness, which is a reflection of that fraction of material capable to become airborne under moderate agitation as comparable to conditions at the workplace (e.g. during packaging operations). Particle size distribution and aerodynamic diameters of the airborne fraction is characterised. The MMAD and GSD of the particles are calculated for with the aid of non-linear regression algorithms. Derived particles distribution parameters are used to quantify the deposited fractions of the airborne dust in the human respiratory tract. This is performed using the Multiple-Path Particle Dosimetry Model (MPPD, v3.04; ARA, 2015) based on morphological data on the human respiratory tract and the aerodynamic diameter of a particle under scrutiny. This model predicts the fraction of inhaled material that is deposited in the extra-thoracic (Head), tracheo-bronchial (TB) and alveolar (PU) regions. The MPPD model algorithms calculate the deposition (and clearance) of mono-disperse and polydisperse aerosols in the respiratory tract for particles ranging from ultra-fine (0.01 microns) to coarse (20 microns) sizes. Within each airway, deposition is calculated using theoretically derived efficiencies for deposition by diffusion, sedimentation and impaction within the airway or airway bifurcation. Filtration of aerosols by the head is determined using empirical efficiency functions.

GLP compliance:

no

Species:

other: humans

Route of administration:

inhalation: aerosol

Details on distribution in tissues:

Based on the MPPD model the following conclusions can be drawn for risk characterisation purposes for all of the tested substances:
(i) the tested Carbon black sample has a limited deposition ability in the human respiratory tract: Only 42.86 % of airborne material is estimated to deposit in the human lung. The rest of the airborne material is not inhaled due to physical phenomena related to air streams and turbulences close to the mouth or simply exhaled (i.e. not deposited).
(ii) about 0.56 % or less of inhaled material are predicted to deposit in the pulmonary region (PU) of the human lung whereas the material deposited in the tracheobronchial (TB) (0.35 %) and the extrathoracic region (Head) may be assumed to be cleared to the GI tract (i.e., by mucociliary escalation and subsequent swallowing).

Dustiness. Particles size distribution, MMAD and GSD

Full description of dustiness testing and results are displayed in section IUCLID 4.28.8. Value for dustiness for Corax N990 is 64.42 mg/g. This represents the fraction of the material that is likely to become airborne upon agitation. The cumulated fractions of the airborne material which deposited at certain stages of the cascade impactor (cut-off points) are given below (Table 2)

Table 2: Cumulated fractions smaller than specific cut-off particle sizes

Cut off point [µm], AD	0.473	0.989	2.04	4.06	8.13	15.8	32.4
CORAX N 990 BDS	0.18%	0.78%	4.16%	6.67%	17.53%	23.04%	41.45%

Table 3 gives the results for the estimated MMADs and GSDs for different model assumptions and regression methods. The results show that approximately 100 % of Carbon black tend to have an MMAD of 49.31 μm.

Test material	p1	MMAD 1	GSD 1
CORAX N 990 BDS	1	49.31	5.72

Prediction of respiratory deposition patterns using the MPPD model

The total deposited fraction of the material that would become airborne under simulated workplace conditions is estimated at 42.86 % for the human worker. The rest of the airborne material would not get inhaled due to physical phenomena related to air streams and turbulences close to the mouth or simply exhaled (i.e. not deposited). When assessing the exposure of specific regions of the respiratory tract, the data indicate that almost all (ca. 42 %) of the deposited material would actually impact the pharynx and is subsequently most likely swallowed. Thus, almost all of the deposited material would contribute to oral and not to inhalation exposure. The deposited fraction in the “TB” region (0.35 %) is also assumed to be discharged into the gastro-intestinal (G) tract by mucociliary escalation and subsequent swallowing. Therefore, the proportion of the airborne material, which may lead to local effects deep in the human lung, reduces to 0,56 % (Table 4, column “PU”) for the tested material.

Table 4: MPPD model outputs using specific input parameters MMAD and GSD estimators

Test material	Head [%]	TB [%]	PU [%]	Total [%]
CORAX N 990 BDS	41.95	0.35	0.56	42.86

Conclusions:

Based on the MPPD model the following conclusions can be drawn for risk characterisation purposes for all of the tested substances:
(i) the tested Carbon black sample has a limited deposition ability in the human respiratory tract: Only 42.86 % of airborne material is estimated to deposit in the human lung. The rest of the airborne material is not inhaled due to physical phenomena related to air streams and turbulences close to the mouth or simply exhaled (i.e. not deposited).
(ii) about 0.56 % or less of inhaled material are predicted to deposit in the pulmonary region (PU) of the human lung whereas the material deposited in the tracheobronchial (TB) (0.35 %) and the extrathoracic region (Head) may be assumed to be cleared to the GI tract (i.e., by mucociliary escalation and subsequent swallowing).

Reference 3

Endpoint:

basic toxicokinetics in vitro / ex vivo

Remarks:

Bioaccessibility

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

September - October 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Objective of study:

bioaccessibility (or bioavailability)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Series on Testing and Assessment No. 29 (23-Jul-2001): Guidance document on transformation/dissolution of metals and metal compounds in aqueous media

Version / remarks:

test medium, loading and test duration adopted for toxicokinetics assessment

Qualifier:

equivalent or similar to guideline

Guideline:

other: the Standard Operating Procedure for Bioelution Testing of Metals, Inorganic Metal Compounds, and Complex Metal-Containing Materials: Simulated Gastric Fluid

Version / remarks:

Version 09.11.2018; submitted by Eurometaux to ECVAM as part of the OECD validation process in November 2018, validated by ECVAM in December 2019

Principles of method if other than guideline:

An internationally agreed guideline does not exist for this test (e.g. OECD). The test was conducted on the basis of the guidance for OECD-Series on testing and assessment Number 29 and according to the bioaccessibility test protocol validated by EURL ECVAM.

The test was performed on the basis of OECD Series on Testing and Assessment No. 29 (2001; ENV/JM/ MONO(2001)9) and the Standard Operating Procedure for Bioelution Testing of Metals, Inorganic Metal Compounds, and Complex Metal-Containing Materials: Simulated Gastric Fluid (version 09.11.2018; submitted by Eurometaux to ECVAM as part of the OECD validation process in November 2018) and was in principle followed. The test media were artificial physiological media: Gamble's solution (GMB) and Artificial gastric fluid (GST).

GLP compliance:

no

Radiolabelling:

no

Species:

other: in vitro (simulated human body fluids)

Details on test animals or test system and environmental conditions:

The objective of this test was to assess the dissolution of carbon black in two artificial physiological media: Artificial gastric fluid (GST, pH 1.5) and Gamble’s solution (GMB, pH 7.4). The test media were selected to simulate relevant human-chemical interactions (as far as practical), e.g. a substance entering the human body by ingestion into the gastro-intestinal tract (GST) or via the respiratory system (GMB).

Test principle in brief:
- two different artificial physiological media: GMB and GST,
- one loading of test substance of 2 g/L,
- the test was performed in triplicates with blanks in quadruplicates
- solutions were sampled after 2 and 24h of agitation (100 rpm) at 37 ± 1 °C; one replicate of GMB was additionally sampled after 168h. Further, the test was performed in a 5 % CO2 atmosphere.
- measurement of dissolved/dispersed carbon concentrations (inorganic carbon and total carbon) after filtration (0.2 µm)
- four additional method blanks per medium

Duration and frequency of treatment / exposure:

Dissolved total carbon (TC), inorganic carbon (IC) and based on indirect determination organic carbon concentrations in GMB and GST media were assessed after 2 and 24 hours of incubation with the test substance. One replicate of GMB solution was analysed after 168 hours to confirm the absence of carbon dissolution.

Dose / conc.:

2 other: g of test item/L artificial media

Details on study design:

Test setup
Four replicate flasks (500 mL glass flasks, one for pH measurement only) per test medium (GMB and GST) were prepared with a loading of 2 g/L. The test item was weighed into flasks, adjusted to volume with the respective artificial physiological medium and agitated at 100 rpm at 37°C ± 1°C. Four control blank replicates (same procedure) per test medium were also prepared.
Four replicates containing the test item (one for pH measurement only) and four method blanks per artificial medium were tested. All solutions were sampled after 2 and 24 h (one replicate of GMB after 168 h.

Dissolved total carbon (TC) and inorganic carbon (IC) were measured by a TOC analyser after 0.2 µm filtration (0.2 µm, Syringe Filter, PET, Chromafil, Macherey-Nagel, Düren, Germany). In addition, temperature, pH and observations, including the appearance of the solution (including colour, turbidity and particle film on the surface) were recorded.

Sample fortification:
In addition, samples of the artificial physiological media were fortified with a known amount of TC and IC (by standard addition of commercial standards) to determine the standard recovery. For detailed information please refer to "Any other information on materials and methods incl. tables".

Reagents:
Purified water (resistivity > 18 MΩ·cm, Pure Lab Ultra water purification system from ELGA LabWater, Celle, Germany)

MgCl2 x 6H2O (extra pure AppliChem, Darmstadt, Germany)
NaCl (p.A., Merck, Darmstadt, Germany)
KCl (p.A., Merck, Darmstadt, Germany)
Na2HPO4 (p.A., Merck, Darmstadt, Germany)
Na2SO4 (p.A., Merck, Darmstadt, Germany)
CaCl2 x 2H2O (p.A., Merck, Darmstadt, Germany)
NaAcetate anhydrous (p.A., Merck, Darmstadt, Germany)
NaHCO3 (p.A., Merck, Darmstadt, Germany)
Na3Citrate x 2H2O (p.A., Merck, Darmstadt, Germany)
HCl 30% (instra-analyzed, plus J.T. Baker, Griesheim, Germany)
HNO3 (Supra, Roth, Karlsruhe, Germany)
NaOH (p.A., Merck, Darmstadt, Germany)

DISSOLVED TOTAL CARBON AND INORGANIC CARBON ANALYSIS
Chemical analysis:
For the measurement of TC and IC a pre-performed and validated calibration was used. Based on the calibration the LOD and LOQ was calculated and reported.
The TC and IC concentrations were determined after 0.2 µm filtration of the samples, whereas the TOC (DOC) was calculated by subtraction of IC from TC.
The measurements were performed according to internal Fraunhofer IME SOPs (Standard Operating Procedures) with a SHIMADZU TOC- V CPH Analyzer.

Analysis by SHIMADZU TOC- V CPH Analyzer
A calculation of the LOD and LOQ according to DIN 32645 by the indirect method (calibration method) was performed with the program SQS (SQS 2013, version 1.00, Software zur statistischen Qualitätskontrolle analytischer Daten; validated software).
Recovery rates of the quality control standards (for TC = Potassium hydrogen phthalate standard solution lot. No. 20029253 and for IC = Sodium hydrogen carbonate standard solution lot. No. 20031922, Bernd Kraft, Duisburg, Germany) and the TC and IC concentrations of ultra-pure water (used a quality control sample to check possible contaminations; measurement of NPOC – standard program for ultra-pure water) were determined.

Details on dosing and sampling:

Total carbon (TC), inorganic carbon (IC) and based on indirect determination organic carbon concentrations in GMB and GST media were assessed after 2 and 24 hours of incubation with 2 g/L test substance. One replicate of GMB solution was analysed after 168 hours to confirm the absence of carbon dissolution.

Type:

other: Bioaccessibility GMB, 2, 24 and 168 h @ 37°C (2 g/L loading, 0.2 µm filtration for phase separation)

Results:

TC, IC & OC concentrations are similar to respective TC, IC & OC background concentrations

Type:

other: Bioaccessibility GST, 2 and 24 h @ 37°C (2 g/L loading, 0.2 µm filtration for phase separation)

Results:

TC, IC & OC concentrations are similar to respective TC, IC & OC background concentrations

Bioaccessibility (or Bioavailability) testing results:

The bioaccessibility of Carbon Black (N990) was determined in vitro by simulating dissolution under physiological conditions in body fluids with a loading of 2 g test item/L.

Total carbon (TC):
Total carbon concentrations of GST method blanks and after 2 and 24h sampling were below the LOQ (LOQ: 1.01 mg TC/L).

Total carbon concentrations of GMB test solutions were above the LOQ (LOQ: 4.27 mg/L) but results were within the calculated range for total carbon background concentration. Hence, concentrations are not distinguishable from background concentrations (i.e. 503 - 525 mg/L).

Inorganic carbon (IC):
Inorganic carbon concentrations of method blanks and GST test solutions were above the LOQ (LOQ: 0.114 mg/L). The concentrations of the test solutions were in the same order of magnitude as concentrations of method blanks and are not distinguishable from background concentrations (i.e. 0.140 - 0.164 mg/L).

Inorganic carbon concentrations of GMB test solutions were above the LOQ (LOQ: 3.90 mg/L) but results were within the calculated range for total carbon background concentration. Hence, concentrations are not distinguishable from background concentrations (i.e. 304 - 325 mg/L).

Organic carbon (OC):
Organic carbon concentrations of GST method blanks and after 2 and 24h sampling were below the LOD/LOQ (same LOD/LOQ as TC).

Inorganic carbon concentration of GMB test solution were determined the LOQ (same LOQ as TC) but results were within the calculated range for organic carbon background concentrations. Hence, concentrations are not distinguishable from background concentrations (i.e. 196 - 200 mg/L).

Table 3: Measured pH of GST method blanks and test solutions.

sample name	target pH	pH prior to the test	pH after 2h	pH after 24h
GST 1	1.5	1.502	Not measured	1.490
GST 2	1.5	1.507		1.481
GST 3	1.5	1.508		1.479
GST 4 (pH measurement vessel)	1.5	1.508	1.480	1.458
GST blank vessel 1	1.5	1.508	1.513	1.520
GST blank vessel 2	1.5	1.507	1.513	1.516
GST blank vessel 3	1.5	1.512	1.521	1.524
GST blank vessel 4	1.5	1.504	1.513	1.512

 

Table 4: Measured pH of GMB method blanks and test solutions.

sample name	target pH	pH prior to the test	pH after 2h	pH after 24h	pH after 7d
GMB 1	7.4	7.397	Not measured	7.484	7.502
GMB 2	7.4	7.413		7.495	7.451
GMB 3	7.4	7.412		7.501	7.473
GMB 4(pH measurement vessel)	7.4	7.396	7.440	7.482	7.456
GMB blank vessel 1	7.4	7.424	7.497	7.487	7.461
GMB blank vessel 2	7.4	7.415	7.491	7.484	7.457
GMB blank vessel 3	7.4	7.420	7.443	7.481	7.460
GMB blank vessel 4	7.4	7.405	7.404	7.483	7.457

 

Test temperature:

With 37 °C ± 1 °C, the temperature was stable during the test for all solutions

 

LOQ/LOD:

Calibration range 0-10 mg/L:

For the total carbon concentration, the LOD is 0.267 mg/L and the LOQ is calculated to 1.01 mg/L. For the inorganic carbon concentration the LOD is 0.029 mg/L and the LOQ is 0.114 mg/L, respectively.

 

Calibration range 10 – 100 mg/L: For the total carbon concentration, the LOD is 1.15 mg/L and the LOQ 4.27 mg/L. For the inorganic carbon concentration the LOD is 1.05 mg/L and the LOQ is 3.90 mg/L, respectively.

 

Recovery:

Table 5: TOC Analyzer measurement series: LOD/LOQ, QA samples and recovery.

Date	Calibration range [mg/L]	Carbon fraction	LOD [mg/L]	LOQ [mg/L]	Quality control standards – after subtraction of mean background concentration ultra-pure water		Ultra-pure water – blank quality control standard; mean concentration measured as NPOC (standard procedure)
September 03, 2020; TC and IC measurements of GST samples 2h	0 - 10 mg/L for all samples	TC	0.267	1.01	1 mg/L: 121 ± 3 (n = 4)	10 mg/L: 108 ± 2 (n = 4)	0.131 ± 0.017 mg/L (n = 8); all concentration below the LOD (LOD of TC used for TOC)
		IC	0.029	0.114	1 mg/L: 129 ± 3 (n = 4)	10 mg/L: 122 ± 11 (n = 4)
September 04, 2020; TC and IC measurements of GST samples 24h	0 - 10 mg/L for all samples	TC	0.267	1.01	1 mg/L: 123 ± 3 (n = 4)	10 mg/L: 108 ± 2 (n = 4)	0.122 ± 0.012 mg/L (n = 8); all concentration below the LOD (LOD of TC used for TOC)
		IC	0.029	0.114	1 mg/L: 122 ± 14 (n = 4)	10 mg/L: 114 ± 19 (n = 4)
September 28, 2020; TC and IC measurements of GMB samples 2h	10 - 100 mg/L for all samples	TC	1.15	4.27	50 mg/L: 99.9 ± 3.0 (n = 4)	100 mg/L: 104 ± 3 (n = 4)	0.217 ± 0.062 mg/L (n = 8); all concentration below the LOD (LOD of TC used for TOC)
		IC	1.05	3.90	50 mg/L: 112 ± 12 (n = 4)	100 mg/L: 107 ± 8 (n = 4)
September 29, 2020; TC and IC measurements of GMB samples 24h	10 - 100 mg/L for all samples	TC	1.15	4.27	50 mg/L: 102 ± 6 (n = 4)	100 mg/L: 106 ± 4 (n = 4)	0.130 ± 0.023 mg/L (n = 8); all concentration below the LOD (LOD of TC used for TOC)
		IC	1.05	3.90	50 mg/L: 124 ± 10 (n = 4)	100 mg/L: 108 ± 13 (n = 4)
October 05, 2020; TC and IC measurements of GMB samples 7d	10 - 100 mg/L for all samples	TC	1.15	4.27	25 mg/L: 92.1 ± 2.8 (n = 4)	50 mg/L: 106 ± 9 (n = 4)	0.199 ± 0.073 mg/L (n = 4); all concentration below the LOD (LOD of TC used for TOC)
		IC	1.05	3.90	25 mg/L: 88.7 ± 1.6 (n = 4)	50 mg/L: 98.8 ± 6.6 (n = 4)

 

The recoveries should be in a range of 100 ± 25% in the lower concentration range (1 mg/L) due to the higher influence of the carbon contamination in ultra-pure water. For higher concentrations (e.g. 10 mg/L) the recoveries should be in the range of 100 ± 20%.

Conclusions:

Measured total carbon, inorganic carbon concentrations and indirectly determined organic carbon concentrations of artificial lung fluid and artificial gastric fluid exposed to 2 g/L Carbon Black (N990) for 2 and 24 h were not different from respective carbon background concentrations of the artificial body fluids. Hence, Carbon Black (N990) is considered non-soluble in artificial lung fluid and artificial gastric fluid.

Executive summary:

The objective of this bioaccessibility study is to investigate the dissolution/dispersion of Carbon Black (N990) (EC 215-609-9; CAS 1333-86-4) in artificial lung fluid and gastric fluid.

The test was performed on the basis of the OECD Series on Testing and Assessment No. 29 (2001; ENV/JM/ MONO(2001)9). The Standard Operating Procedure for Bioelution Testing of Metals, Inorganic Metal Compounds, and Complex Metal-Containing Materials: Simulated Gastric Fluid (validated by ECVAM in December 2019) was followed in principle .

The test media were selected to simulate relevant human-chemical interactions of the substance by entering the human body by ingestion into the gastro-intestinal tract (GST) or by inhalation via the respiratory system (GMB).

The test solutions with a loading of 2g/L (triplicates and one additional flask for pH measurements) were agitated at 100 rpm at 37°C ± 1°C. Test solutions were sampled for analysis after 2 h, 24 h and one GMB replicate after 168 h.

Dissolved total carbon concentrations (TC) and inorganic carbon concentrations (IC) were measured with a SHIMADZU TOC- V CPH Analyzer after filtration (0.2 µm, Syringe Filter, PET, Chromafil, Macherey-Nagel, Düren, Germany). Organic carbon concentrations were determined indirectly by subtraction (OC=TC-IC). In addition, temperature, pH and observations, including the appearance of the solution (including colour, turbidity and particle film on the surface) were recorded.

Measured total carbon, inorganic carbon concentrations and indirectly determined organic carbon concentrations of artificial lung fluid and artificial gastric fluid exposed to 2 g/L Carbon Black (N990) for 2 and 24 h were not different from respective carbon background concentrations of the artificial body fluids. Hence, Carbon Black (N990) is considered insoluble in artificial lung fluid and artificial gastric fluid.

Description of key information

See discussion under key information for set_not treated

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

0

Absorption rate - dermal (%):

0

Absorption rate - inhalation (%):

0

Additional information

Results from bioelution/solubility testing in artificial biological fluids [Artificial gastric fluid (GST, pH 1.5) mimicking the very harsh digestion milieu of high acidity in the stomach (ASTM D5517; (Stopford et al. 2003)], performed on the basis of the OECD Series on Testing and Assessment No. 29 (2001; ENV/JM/ MONO(2001)9) and following the Standard Operating Procedure for Bioelution Testing of Metals, Inorganic Metal Compounds, and Complex Metal-Containing Materials: Simulated Gastric Fluid (validated by ECVAM in December 2019) show that untreated nanoforms of carbon black are insoluble in GST. CORAX N990 was insoluble after a 2 and 24-hour incubation in GST. Similarly, no solubility, measured as total carbon, inorganic carbon concentrations and indirectly determined organic carbon concentrations was recorded for the same grades in artificial interstitial fluid (Gamble’s solution (GMB, pH 7.4) mimicking conditions within the deep lung under normal health conditions (Midander et al. 2007). After 2 hours, 24 hours and 7 days incubation in the GMB, values for total and inorganic carbon were not different from the background concentrations.